Chapter 17

FromGto Prot

KEY CONCEPTS

n

... Figure 17.1 How does a single faulty gene result in thedramatic appearance of an albino deer?

17.1 Genes specify proteins via transcription andtranslation

17.2 Transcription is the DNA·directed synthesis ofRNA: a closer look

17.3 Eukaryotic cells modify RNA after transcription17.4 Translation is the RNA-directed synthesis of a

polypeptide: a closer look

17.5 Point mutations can affect protein structureand function

17.6 While gene expression differs among the domainsof life, the concept of a gene is universal

r;;::;·~~::·of Genetic Information

I n 2006, a young albino deer seen frolicking with severalbrown deer in the mountains of eastern Germanyelicited a public outcry (Figure 17.1). A local hunting or

ganization said the albino deer suffered from a "genetic disorder~ and should be shot. Some people felt the deer shouldmerely be prevented from mating with other deer in order tosafeguard the gene pool of the population. Others favored re

locating the albino deer to a nature reserve because theyworried that it might be more noticeable to predators if leftin the wild. A German rock star even held a benefit concert

to raise funds for the relocation. What led to the strikingphenotype of this deer, the cause of this lively debate?

You learned in Chapter 14 that inherited traits are determined by genes and that the trait of albinism is caused by arecessive allele ofa pigmentation gene. The information content of genes is in the form of specific sequences of nucleotides along strands of DNA, the genetic material. But howdoes this information determine an organism's traits? Putanother way, what does a gene actually say? And how is its

message translated by cells into a specific trait, such as brownhair, type A blood, or, in the case of an albino deer, a totallack of pigment? The albino deer has a faulty version of a keyprotein, an enzyme required for pigment synthesis, and thisprotein is faulty because the gene that codes for it contains

incorrect information.This example illustrates the main point ofthis chapter: The

DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins and of RNA molecules involved in protein synthesis. In other words, proteins are thelink between genotype and phenotype. Gene expression isthe process by which DNA directs the synthesis of proteins(or, in some cases, just RNAs). The expression of genes thatcode for proteins includes two stages: transcription and translation. This chapter describes the flow of information fromgene to protein in detail and explains how genetic mutationsaffect organisms through their proteins. Gene expression involves similar processes in all three domains of life. Under

standing these processes will allow us to revisit the concept ofthe gene in more detail at the end of the chapter.

r~:~:s'::~f: proteins viatranscription and translation

Before going into the details of how genes direct protein synthesis, let's step back and examine how the fundamental relationship between genes and proteins was discovered.

Evidence from the Study of Metabolic DefectsIn 1909, British physician Archibald Garrod was the first tosuggest that genes dictate phenotypes through enzymes that

325

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catalyze specific chemical reactions in the cell. Garrod postulated that the symptoms of an inherited disease reflect a person's inability to make a particular enzyme. He referred tosuch diseases as "inborn errors of metabolism." Garrod gave

as one example the hereditary condition called alkaptonuria,

in which the urine is black because it contains the chemicalalkapton, which darkens upon exposure to air. Garrod reasoned that most people have an enzyme that metabolizes alkapton, whereas people with alkaptonuria have inherited aninability to make that enzyme.

Garrod may have been the first person to recognize thatMendel's principles of heredity apply to humans as well aspeas. Garrod's realization was ahead of its time, but researchconducted several decades later supported his hypothesisthat a gene dictates the production ofa specific enzyme. Biochemists accumulated much evidence that cells synthesizeand degrade most organic molecules via metabolic pathways,in which each chemical reaction in a sequence is catalyzed by

a specific enzyme (see p. 142). Such metabolic pathways lead,for instance, to the synthesis of the pigments that give fruitflies (Drosophila) their eye color (see Figure 15.3). In the

193Os, George Beadle and Boris Ephrussi speculated that inDrosophila, each of the various mutations affecting eye colorblocks pigment synthesis at a specific step by preventing production of the enzyme that catalyzes that step. However, neither the chemical reactions nor the enzymes that catalyzethem were known at the time.

Nutritional Mutants in Neurospora: Scientific Inquiry

A breakthrough in demonstrating the relationship betweengenes and enzymes came a few years later, when Beadle and

Edward Tatum began working with a bread mold, Neurosporacrassa. Using a treatment shown in the 1920s to cause geneticchanges, they bombarded Neurospora with X-rays and thenlooked among the survivors for mutants that differed in theirnutritional needs from the wild-type mold. \Vild-typeNeurospora has modest food requirements. It can survive inthe laboratory on a moist support medium called agar, mixedonly with inorganic salts, glucose, and the vitamin biotin.From this minimal medium, the mold cells use their metabolic pathways to produce all the other molecules they need.Beadle and Tatum identified mutants that could not surviveon minimal medium, apparently because they were unable to

synthesize certain essential molecules from the minimal ingredients. To ensure survival of these nutritional mutants,Beadle and Tatum allowed them to grow on a completegrowthmedium, which consisted of minimal medium supplementedwith all 20 amino acids and a few other nutrients. The complete growth medium could support any mutant that couldn'tsynthesize one of the supplements.

To characterize the metabolic defect in each nutritionalmutant, Beadle and Tatum took samples from the mutant

326 UNIT THREE Genetics

growing on complete medium and distributed them to a number of different vials. Each vial contained minimal mediumplus a single additional nutrient. The particular supplementthat allowed growth indicated the metabolic defect. For ex

ample, if the only supplemented vial that supported growth of

the mutant was the one fortified with the amino acid arginine,the researchers could conclude that the mutant was defective

in the biochemical pathway that wild-type cells use to synthesize arginine.

Beadle and Tatum went on to pin down each mutant'sdefect more specifically. Figure 17.2 shows how they used additional tests to distinguish among three classes of argininerequiring mutants. Mutants in each class required a differentset of compounds along the arginine-synthesizing pathway,which has three steps. Based on their results, the researchersreasoned that each class must be blocked at a different step inthis pathway because mutants in that class lacked the enzymethat catalyzes the blocked step.

Because each mutant was defective in a single gene, Beadleand Tatum's results provided strong support for the onegene-one enzyme hypothesis, as they dubbed it, which statesthat the function ofa gene is to dictate the production ofa specific enzyme. Further support for this hypothesis came fromexperiments that identified the specific enzymes lacking in themutants. Beadle and Tatum shared a Nobel Prize in 1958 for"their discovery that genes act by regulating definite chemicalevents" (in the words of the Nobel committee).

The Products of Gene Expression: A Developing Story

As researchers learned more about proteins, they made revisions to the one gene-one enzyme hypothesis. First of all, not

all proteins are enzymes. Keratin, the structural protein of animal hair, and the hormone insulin are two examples of nonenzyme proteins. Because proteins that are not enzymes arenevertheless gene products, molecular biologists began tothink in terms of one gene-one protein. However, many proteins are constructed from two or more different polypeptidechains, and each polypeptide is specified by its own gene. Forexample, hemoglobin, the oxygen-transporting protein of vertebrate red blood cells, is built from two kinds ofpolypeptides,and thus tv.·o genes code for this protein (see Figure 5.21).

Beadle and Tatum's idea was therefore restated as the onegene-one polypeptide hypothesis. Even this description is not

entirely accurate, though. First, many eukaryotic genes cancode for a set ofclosely related polypeptides in a process calledalternative splicing, which you will learn about later in thischapter. Second, quite a few genes code for RNA moleculesthat have important functions in cells even though they arenever translated into protein. For now, we will focus on genesthat do code for polypeptides. (Note that it is common to referto these gene products as proteins, rather than more preciselyas polypeptides-a practice you will encounter in this book.)

In ui• FI~11.2

Do individual genes specify the enzymes that functionin a biochemical pathway?

EXPERIMENT Working with the mold Neurospora craS5il, George Beadle and EdwardTatum, then at Stanford University, isolated mutants that required arginine in their growthmedium. The researchers showed that these mutants fell into three classes, each defectivein adifferent gene. From other considerations, they suspected that the metabolic pathwayof arginine biosynthesis involved aprecursor nutrient and the intermediate moleculesornithine and citrulline, Their most famous experiment, shown here, tested both their onegene-one enzyme hypothesIs and their postulated arginine-synthesizing pathway. In thisexperiment, they grew their three classes of mutants under the four different conditionsshown in the Results section below. They included minimal medium (MM) as a controlbecause they knew that wild-type cells could grow on MM but mutant cells could not. (Seetest tubes on the right.)

Growth: ---1'1Wild-typecells growingand dividing

Minimal medium

No growth:Mutant cellscannot growand divide

SOURCE

Classes of Neurospora crassa

RESULTS The Wild-type strain was Wild type Class I mutants Class II mutants Class III mutantscapable of growth under all experimental Minimal

~ ~ ~ ~conditions, requiring only the minimal mediummedium. The three classes of mutants each (MM)had a specific set of growth requirements, (control)For example, class II mutants could not

~ ~ ~ ~grow when ornithine alone was added but

MM'could grow when either citrulline or < ornithinearginine was added, 0~

"<

~ ~ ~ ~0u MM,

citrulline

MM,

~ ~ ~ ~arginine(control)

Can grow with or Can grow on Can grow only on Absolutely requirewithout any ornithine, citrulline, citrulline or arginine to growsupplements or arginine arginine

CONCLUSION From the growth Class J mutants Class II mutants Class III mutantsrequirements of the mutants, Beadle and (mutation in (mutation in (mutation inTatum deduced that each class of mutant Wild type gene A) gene B) gene C)was unable to carry out one step in thepathway for synthesizing arginine, Precursor Precursor Precursor Precursorpresumably because it lacked the necessary

Gene A - '-' 'X' '-' '-'enzyme, Because each of their mutants wasmutated in asingle gene, they concluded

Ornithine Ornithine Ornithine Ornithinethat each mutated gene must normallydictate the production of one enzyme, Their Gene B - E1>:yme B '-" 'X' E1xy..... B

results supported the one gene-one enzymehypothesiS and also confirmed the arginine Citrulline Citrulline Citrulline Citrullinepathway, (Notice in the Results that amutant Gene C_ '-' Enzym. C Eo...,...,. C 'X'can grow only if supplied with a compoundmade after the defective step, because this Arginine Arginine Arginine Argininebypasses the defect.)

G W Be~dle ~nd E L hlum. Genellc control of bi<xhemic~1 react,OI1s in Nel.lr~~,ProceedingS of rhe N~rl(j(l~1 Academyof Xl/'!fICeS 27:499-506 (1941)

-I/@il i• Suppose the experiment had shown that class I mutants could grow only in MMsupplemented by ornithine or arginine and that class II mutants could grow in MM supplementedby citrulline, ornithine, or arginine. What conclusions would Beadle and Tatum have drawn fromthose results regarding the biochemical pathway and the defect in class [and class II mutants?

(~APTE~ SEVENTEEN From Gene to Protein 327

Basic Principles ofTranscriptionand Translatoon

Genes provide the instructions for making specific proteins.

But a gene does not build a protein directly. The bridge between DNA and protein synthesis is the nucleic add RNA.You learned in Chapter 5 that RNA is chemically similar toDNA, except that it contains ribose instead ofdeoxyribose asits sugar and has the nitrogenous base uracil rather thanthymine (see Figure 5.27). Thus, each nucleotidealong a DNAstrand has A. G. C, or T as its base. and each nucleotide alongan RNA strand has A, G, C. or U as its base. An RNA molecule llSuaUy consists ofa single strand.

It is customary to describe the flow of information fromgene to protein in linguistic terms because both nucleic acidsand proteins are polymers with specific sequences ofmonomers that convey information, much as specific se

quences of letters communicate information in a languagelike English. In DNA or RNA, the monomers are the fourtypes of nucleotides, which differ in their nitrogenous bases.Genes are typically hundreds or thousands of nucleotideslong, each gene having a specific sequence of bases. Eachpolypeptide of a protein also has monomers arranged in aparticular linear order (the protein's primary structure), butits monomers are amino adds. Thus, nucleic adds and proteins contain information written in two different chemicallanguages. Getting from DNA to protein requires two majorstages: transcription and translation.

Transcription is the synthesis of RNA under the direction of DNA. Both nucleic acids use the same language, and

the information is simply transcribed, or copied, from onemolecule to the other. Just as a DNA strand provides atemplate for the synthesis of a new complementary strandduring DNA replication, it also can serve as a template forassembling a complementary sequence of RNA nucleotides.For a protein-coding gene, the resulting RNA molecule is afaithful transcript of the gene's protein-building instructions, in the same way that your college transcript is an accurate record of your grades, and like a transcript, it can besent out in multiple copies. This type of RNA molecule iscalled messenger RNA (mRNA) because it carries a geneticmessage from the DNA to the protein-synthesizing machinery of the cell. (Transcription is the general term for the

synthesis of any kind of RNA on a DNA template. Later inthis chapter, you will learn about some other types of RNAproduced by transcription.)

Translation is the synthesis ofa polypeptide, which occursunder the direction of mRNA. During this stage, there is achange in language: The cell must translate the base sequenceof an mRNA molecule into the amino acid sequence of apolypeptide. The sites of translation are ribosomes, complexparticles that facilitate the orderly linking of amino acids into

polypeptide chains.

328 UNIT TUII Genetics

Transcription and translation occur in all organisms. Recallfrom Chapter I that there are three domains of life: Bacteria,

Archaea, and Eukarya. Organisms in the first two domains aregrouped as prokaryotes because their cells lack a membrane

bounded nucleus-a defining feature ofeukaryotic cells. Moststudies of transcription and translation have been done onbacteria and eukaryotes, which are therefore our main focus inthis chapter. Although our understanding of these processesin archaea lags behind, in the last section we will discuss a fewaspects of archaeal gene expression.

The basic mechanics of transcription and translation aresimilar for bacteria and eukaryotes, but there is an importantdifference in the flow ofgenetic information within the cells.Because bacteria do not have nuclei, their DNA is not segregated from ribosomes and the other protein-synthesizingequipment (Figure 17.3a). As you will see later, this lack of

segregation allows translation of an mRNA to begin whileits transcription is still in progress. In a eukaryotic cell, bycontrast, the nuclear envelope separates transcriptionfrom translation in space and time (Figure 11.3b). Transcription occurs in the nucleus, and mRNA is transportedto the cytoplasm, where translation occurs. But beforethey can leave the nucleus, eukaryotic RNA transcriptsfrom protein-coding genes are modified in various ways toproduce the final, functional mRNA. The transcription ofa protein-coding eukaryotic gene results in pre-mRNA,and further processing yields the finished mRNA. The ini

tial RNA transcript from any gene, including those codingfor RNA that is not translated into protein, is more gener

ally called a primary transcript.Let's summarize: Genes program protein synthesis via ge

netic messages in the form of messenger RNA. Put anotherway, cells are governed by a molecular chain ofcommand witha directional flow ofgenetic information: DNA ---+ RNA ---+ protein. This concept was dubbed the central dogma by FrancisCrick in 1956. How has the concept held up over time? In the19705, scientists were surprised to discover that some RNAmolecules can act as templates for DNA, a process you1l readabout in Chapter 19. However, this rare exception does not invalidate the idea that, in general, genetic information flowsfrom DNA to RNA to protein. In the next section, we discuss

how the instructions for assembling amino acids intoa specificorder are encoded in nucleic acids.

The Genetic Code

When biologists began to suspect that the instructions forprotein synthesis were encoded in DNA, they recognized aproblem: There arc only four nucleotide bases to specify 20amino acids. Thus, the genetic code cannot be a languagelike Chinese, where each written symbol corresponds to aword. How many bases, then, correspond to an amino

acid?

3'

3'

S'

jjj

Gene 2

Codon

j

Amino acid

S'

DNA~mo'"," ~Gene 1

:\7\

in each position, this would give us 16 (that is, 42) possible

arrangements-still not enough to code for all 20 amino acids.

Triplets of nucleotide bases are the smallest units of uniform length that can code for all the amino acids. If each

arrangement of three consecutive bases specifies an aminoacid, there can be 64 (that is, 43) possible code words-more

than enough to specify all the amino acids. Experiments haveverified that the flow of information from gene to protein isbased on a triplet code: TIle genetic instructions for apolypeptide chain are written in the DNA as a series ofnonoverlapping, three-nucleotide words. For example, thebase triplet AGT at a particular position along a DNA strandresults in the placement of the amino acid serine at the corresponding position of the polypeptide being produced.

During transcription, the gene determines the sequence ofbases along the length of an mRNA molecule (Figure 17.4).

For each gene, only one ofthe two DNA strands is transcribed.This strand is called the template strand because it providesthe pattern, or template, for the sequence of nucleotides in anRNA transcript. Agiven DNA strand is the template strand forsome genes along a DNA molecule, while for other genes thecomplementary strand functions as the template. Note that

DNAtemplate J 3'strand~

TRANSLATION

Protein

mRNA

// // // // //ITRANSCRIPTION I IDNA

~

I I IPre-mRNARNA PROCESSING

~~

mRNA

Codons: Triplets of Bases

(al Bacterial cell. In a badenal cell, whICh lacks anucleus, mRNA produced by transcription isimmediately translated without additional processing.

.. Figure 17.3 Overview: the roles of traMCl'iption andtranslation in the flow of genetic information. In a cell. inhernedinformation flows from DNA to RNA to protein. The two main stages ofinformation flow are transcription and translation. A miniature version ofpart (a) or (b) accompanies several figures later in tile cilapter as anorientation diagram to ilelp you see wilere a particular figure fits into tileoverall scileme

(b) Eukaryotic cell. The nucleus provides a separatecompartment for transcription. The original RNAtranscript, called pre-mRNA. is processed in variousways before leaving the nucleus as mRNA.

r",'",'",'",'// .'/ .'/ .'/ .'/riRANSCRI'iJON I j DNA

~! mRNA..~s:meI TRANSLATION I ~.;,,~Polypeptide

I TRANSLATION I Ribosome

~,~

If each nucleotide base were translated into an amino acid,

only 4 of the 20 amino acids could be specified. Would a language of two-letter code words suffice? The two-base se

quence AG, for example, could specify one amino acid, andGT could specify another. Since there are four possible bases

... Figure 17.4 The triplet code. For each gene, one DNA strandfunctions as a template for transcription. The base-pairing rules for DNAsynthesis also guide transcription, but uracil (U) takes the place of thymine(T) in RNA. During translation, the mRNA is read as a sequence of basetriplets. called codons. Each codon specifies an amino acid to be added tothe growing polypeptide chain. The mRNA is read in the 5' > 3' direction.

C~APTE~ SEVENTEEN From Gene to Protein 329

Second mRNA base

UUU J UAU J UGU JPh' UA( Tyr C"UUC UGC

UUA J UAA Stop UGA Stopl,"

UUG UAG Stop UGG T,p

C C0

CCU ](AU J

CGU]

0~ ~

0 His 0u cec CAC CGC

u

'0 -Pm CGA Arg0

~ ceACAA]

~

c Glo c• •10 ceG CAG CGG

'"•ACU ]

AGUJ•

AAU J•• •• ,",0 S" ~

~

< Ace AAC AGC <z Th' z= ACA AM] AGA] =E Ly, A<g E• ACG MG AGG ~• ~~ •

GCU ] GAU] GGU]GCC

A,pGA( GGC

G(A Ala GAAJ GGA GlyGlc

GCG GAG GGG

Although more elaborate techniques were required to decode mixed triplets such as AVA and CGA, al164 codons weredeciphered by the mid-1960s. As Figure 17.5 shows,61 ofthe64 triplets code for amino acids. The three codons that do notdesignate amino acids are "stop" signals, or terminationcodons, marking the end of translation. Notice that the codonAVG has a dual function: It codes for the amino acid methionine (Met) and also functions as a "start" signal, or initiationcodon. Genetic messages begin with the mRNA codon AVG,which signals the protein~synthesizingmachinery to begin

translating the mRNA at that location. (Because AUG alsostands for methionine, polypeptide chains begin with methionine when they are synthesized. However, an enzyme maysubsequently remove this starter amino acid from the chain.)

Notice in Figure 17.5 that there is redundancy in the geneticcode, but no ambiguity. For example, although codons GAAand GAG both specify glutamic acid (redundancy), neither ofthem ever specifies any other amino acid (no ambiguity). Theredundancy in the code is not altogether random. In manycases, codons that are synonyms for a particular amino aciddiffer only in the third base of the triplet. We will consider apossible benefit of this redundancy later in the chapter.

.... Figure 17.5 The dictionary of the genetic code. The threebases of an mRNA codon are designated here as the first, second, andthird bases, reading in the 5' • 3' direction along the mRNA. (Practiceusing this dictionary by finding the codons in Figure 17.4.) The codonAUG not only stands for the amino acid methionine (Met) but alsofunctions as a "start" signal for ribosomes to begin translating themRNA at that point. Three of the 64 codons function as "stop"signals, marking the end of a genetic message. 5ee Figure 5,17 for alist of the three-letter abbreviations for all the amino acids,

for a particular gene, the same strand is used as the templateevery time it is transcribed.

An mRNA molecule is complementary rather than identi~

cal to its DNA template because RNA bases are assembled on

the template according to base~pairing rules. The pairs are

similar to those that form during DNA replication, except thatU, the RNA substitute for T, pairs with A and the mRNA nu

cleotides contain ribose instead of deoxyribose. Like a newstrand of DNA, the RNA molecule is synthesized in an anti

parallel direction to the template strand of DNA. (To reviewwhat is meant by ~antiparallel" and the 5' and 3' ends of a nu

cleic acid chain, see Figure 16.7.) For example, the base tripletACC along the DNA (written as 3'-ACC-5') provides a template for 5'-UGG-3' in the mRNA molecule. The mRNA basetriplets are called codons, and they are customarily written inthe 5' -~ 3' direction. In our example, UGG is the codon for theamino acid tryptophan (abbreviated Trp). The term codon isalso used for the DNA base triplets along the nontemplatestrand. These codons are complementary to the templatestrand and thus identical in sequence to the mRNA exceptthat they have T instead of U. (For this reason, the nontemplate DNA strand is sometimes called the ~coding strand.~)

During translation, the sequence ofcodons along an mRNAmolecule is decoded, or translated, into a sequence of amino

acids making up a polypeptide chain. The codons are read bythe translation machinery in the 5' ---+ 3' direction along the

mRNA. Each codon specifies which one of the 20 amino acidswill be incorporated at the corresponding position along apolypeptide. Because codons are base triplets, the number ofnucleotides making up a genetic message must be three timesthe number of amino acids in the protein product. For exam

ple, it takes 300 nucleotides along an mRNA strand to code forthe amino acids in a polypeptide that is 100 amino acids long.

Cracking the Code

Molecular biologists cracked the code of life in the early 1960swhen a series of elegant experiments disclosed the amino acidtranslations of each of the RNA codons. The first codon wasdeciphered in 1961 by Marshall Nirenberg, ofthe National In~

stitutes of Health, and his colleagues. Nirenberg synthesized

an artificial mRNA by linking identical RNA nucleotides containing uracil as their base. No matter where this messagestarted or stopped, it could contain only one codon in repetition: VVV. Nirenberg added this ~poly-V~ to a test-tube mixture containing amino acids, ribosomes, and the other

components required for protein synthesis. His artificial system translated the poly-V into a polypeptide containing manyunits of the amino acid phenylalanine (Phe), strung togetheras a long polyphenylalanine chain. Thus, Nirenberg determined that the mRNA codon UUV specifies the amino acidphenylalanine. Soon, the amino acids specified by the codonsAAA, GGG, and CCC were also determined.


Our ability to extract the intended message from a writtenlanguage depends on reading the symbols in the correctgroupings-that is, in the correct reading frame. Considerthis statement: "The red dog ate the bug.n Group the letters in

correctly by starting at the wrong point, and the result willprobably be gibberish: for example, aher edd oga tet heb ug.n

The reading frame is also important in the molecular languageofcells. The short stretch of polypeptide shown in Figure 17.4,for instance, will be made correctly only if the mRNA nudeotides are read from left to right (5' ---> 3') in the groups of

three shown in the figure:.llii.G..!1!.!!.! GQ.C!.KA. Although agenetic message is written with no spaces between the codons,the cell's protein-synthesizing machinery reads the message asa series of nonoverlapping three-letter words. The message isnot read as a series of overlapping words-llG.G.UUU, and soon-which would convey a very different message.

Evolution of the Genetic Code

have produced many exciting developments in the area ofbiotechnology (see Chapter 20).

Exceptions to the universality of the genetic code includetranslation systems in which a few codons differ from the stan

dard ones. Slight variations in the genetic code exist in certainunicellular eukaryotes and in the organelle genes of somespecies. There are also exceptions in which stop codons can betranslated into one of two amino acids not found in most organisms. Although one of these amino acids (pyrrolysine) hasbeen detected thus far only in archaea, the other (selenocysteine) is a component ofsome bacterial proteins and even somehuman enzymes. Despitetheseexceptions, the evolutionarysignificance of the code's near universality is dear. A languageshared by all living things must have been operating very earlyin the history oflife-early enough to be present in the commonancestor of all present·day organisms. A shared genetic vocabulary is a reminder of the kinship that bonds all life on Earth.

... Figure 17.6 Expression of genes from different species.Because di~erse forms of life share a common genetic code. one speciescan be programmed to produce proteins characteristic of asecondspecies by introducing DNA from the second species into the first.

The genetic code is nearly universal, shared by organismsfrom the simplest bacteria to the most complex plants andanimals. The RNA codon CCG, for instance, is translated asthe amino acid proline in all organisms whose genetic codehas been examined. In laboratory experiments, genes can be

transcribed and translated after being transplanted fromone species to another, sometimes with quite striking results, as shown in Figure 17.6! Bacteria can be programmedby the insertion of human genes to synthesize certain humanproteins for medical use, such as insulin. Such applications

CONCEPT CHECK 17.11. What polypeptide product would you expect from a

poly-G mRNA that is 30 nudeotides long?2.••p.W"1 The template strand of a gene contains

the sequence 3'-TTCAGTCGT-5'. Draw the nontemplate sequence and the mRNA sequence, indicating 5'and 3' ends of each. Compare the two sequences.

3. _','!l0'1,. Imagine that the nontemplate sequencein question 2 was transcribed instead of the template

sequence. Draw the mRNA sequence and translate itusing Figure 17.5. (Be sure to pay attention to the 5'and 3' ends.) Predict how well the protein synthesized

from the nontemplate strand would function, if at alL

For suggested answers, see Appendix A.

Molecular Components ofTranscription

Now that we have considered the linguistic logic and evolutionary significance of the genetic code, we are ready to reexamine transcription, the first stage of gene expression, inmore detail.

r;~:~sjc'r~p~~~~s theDNA-directed synthesis of RNA:a closer look

Messenger RNA, the carrier of information from DNA tothe cell's protein-synthesizing machinery, is transcribedfrom the template strand of a gene. An enzyme called anRNA polymerase pries the two strands of DNA apart andjoins the RNA nucleotides as they base-pair along the DNA

(b) Pig expressing a jellyfishgene. Researchers injected thegene for a fluorescent proteminto fertilized pig eggs Oneof the eggs developed intothis fluorescent pig.

(a) Tobacco plant expressing afirefly gene. The yellow glowis produced by a chemicalreadion catalyzed by theprotein produd of the fireflygene.

(~APH~ SEVENTEEN From Gene to Protein 331

template (Figure 17.7). Like the DNA polymerases thatfunction in DNA replication, RNA polymerases can assemble a polynucleotide only in its 5' -. 3' direction. UnlikeDNA polymerases, however, RNA polymerases are able tostart a chain from scratch; they don't need a primer.

Spedfic sequences of nucleotides along the DNA markwhere transcription ofa gene begins and ends. The DNA se·quence where RNA polymerase attaches and initiates tran·scription is known as the promoter; in bacteria, the sequencethat signals the end of transcription is called the terminator.(The termination mechanism is different in eukaryotes; we'lldescribe it later.) Molecular biologists refer to the direction oftranscription as ~downstream" and the other direction as "up_stream." These terms are also used to describe the positions ofnucleotide sequences within the DNA or RNA. Thus, the pro-

moter sequence in DNA is said to be upstream from the terminator. The stretch of DNA that is transcribed into an RNAmolecule is called a transcription unit.

Bacteria have a single type of RNA polymerase that synthesizes not only mRNA but also other types of RNA thatfunction in protein synthesis, such as ribosomal RNA. In con·trast, eukaryotes have at least three types of RNA polymerasein their nuclei. The one used for mRNA synthesis is calledRNA polymerase II. The other RNA polymerases transcribeRNA molecules that are not translated into protein. In the discussion of transcription that follows, we start with the featuresof mRNA synthesis common to both bacteria and eukaryotesand then describe some key differences.

Synthesis of an RNA Transcript

The three stages of transcription, as shown in Figure 17.7 anddescribed next, are initiation, elongation, and termination ofthe RNA chain. Study Figure 17.7 to familiarize yourself withthe stages and the terms used to describe them.

RNA Polymerase Binding and Initiationof Transcription

The promoter of a gene includes within it the transcriptionstart point (the nucleotide where RNA synthesis actually begins) and typically extends several dozen nucleotide pairs

o Initiation. After RNA polymerasebinds to the promoter, the DNAstrands unwind. and the polymeraseinitiates RNA synthesIs at the startpoint on the template strand.

Transcription unit,----'----

Promoter

Start point

RNA polymerase

=(:=~~::::::;;;::===:=_ 3'~ 5'DNA

5'3'

Direction of transcription("downstream") Template

strand of DNA

•

RNApolymerase

Nontemplatestrand of DNA.------...

Elongation

5'

f) Termination. Eventually. the RNAtranscript is released. and thepolymerase detaches from the DNA.

f) Elongation. The polymerase movesdownstream. unwinding the DNA andelongating the RNA transcript 5' -; 3'.In the wake of transcription, the DNA 3'strands re-form a double helix.

RNAtranscript

RewoundD~ 5'

~~~=3'5'5'

~,,~=:\~~==::;:i==~~l:j_....\ RNA Template strand

Unwound transcript of DNADNA

5'3'

5'3'

5'3'~===~_3'= 5'

S'_....~~~ 3'Completed RNA transcript

.. Figure 17.7 The stages of transcription: initiation,elongation. and termination. This general depiction oftranscription applies to both bacteria and eukaryotes. but the details oftermination differ. as described in the te)(!. Also. in a bacterium. the RNAtranscript is immediately usable as mRNA; in a eukaryote. the RNAtranscript must first undergo processing.


Transcription initiation complex

= "5'

RNA transcript

==="5'

RNA polymerase II

~

o A eukaryotk promotercommonly includes a TATAbox. a nucleotide sequencecontaining TATA. about 25nucleotides upstream from

[~~~r:t;; the transcriptional start

_ point. (By convention,lAANS""O:«~ nucleotide sequences are

"" given as they occur on the'-------'::.:.-"'promoter nontemplate strand.)

~:~~~~m~;;~~'~!TATA box Start point

5'_3'===-

"~5'

TemplateDNA strand

~e Several transcription

factors, one recognizingthe TATA box. must bindto the DNA before RNA

Transcription polymerase II can do so

;~~

(an RNA sequence) functions as the termination signal, caus

ing the polymerase to detach from the DNA and release thetranscript, which is available for immediate use as mRNA. Ineukaryotes, RNA polymerase II transcribes a sequence on theDNA called the polyadenylation signal sequence, which codesfor a polyadenylation signal (AAUAAA) in the pre-mRNA.Then, at a point about 10 to 35 nucleotides downstream from

oAdditional transcriptionfactors (purple) bind tothe DNA along with RNApolymerase II. forming thetranSCription initiationcomplex. The DNA doublehelix then unwinds. andRNA synthesis begins atthe start point on thetemplate strand

... Figure 17.8 The initiation of transcription at aeukaryotic promoter. In eukaryotic cells. proteins called transcriptionfactors mediate the initiation of transcription by RNA polymerase II.

D Explain how the interaction of RNA polymerase with the promoterwould differ jf the figure showed transcription initiation for bacteria.

Termination of Transcription

TIle mechanism of termination differs between bacteria andeukaryotes. In bacteria, transcription proceeds through a terminator sequence in the DNA. The transcribed terminator

Elongation of the RNA Strand

upstream from the start point. In addition to serving as abinding site for RNA polymerase and determining wheretranscription starts, the promoter determines which of thetwo strands of the DNA helix is used as the template.

Certain sections of a promoter are especially important for

binding RNA polymerase. In bacteria, the RNA polymeraseitselfspecifically recognizes and binds to the promoter. In eukaryotes, a collection of proteins called transcription factorsmediate the binding of RNA polymerase and the initiation oftranscription. Recall from Chapter 16 that the DNA ofa eukaryotic chromosome is complexed with histones and other proteinsin the form of chromatin. The roles of these proteins in makingthe DNA accessible to transcription factors will be discussed inChapter 18. Only after certain transcription factors are attachedto the promoter does RNA polymerase II bind to it. The wholecomplex of transcription factors and RNA polymerase II boundto the promoter is called a transcription initiation complex.Figure 17.8 shows the role of transcription factors and a crucialpromoter DNA sequence called a TATA box in forming the ini

tiation complex at a eukaryotic promoter.The interaction between eukaryotic RNA polymerase II

and transcription factors is an example of the importance ofprotein-protein interactions in controlling eukaryotic transcription. Once the polymerase is firmly attached to the promoter DNA, the two DNA strands unwind there, and the

enzyme starts transcribing the template strand.

As RNA polymerase moves along the DNA, it continues tountwist the double helix, exposing about 10 to 20 DNA basesat a time for pairing with RNA nudeotides (see Figure 17.7).The enzyme adds nudeotides to the 3' end of the growingRNA molecule as it continues along the double helix. In thewake of this advancing wave of RNA synthesis, the new RNAmolecule peels away from its DNA template and the DNAdouble helix re·forms. Transcription progresses at a rate of

about 40 nudeotides per second in eukaryotes.A single gene can be transcribed simultaneously by several

molecules of RNA polymerase following each other like trucksin a convoy. A growing strand of RNA trails off from eachpolymerase, with the length of each new strand reflecting howfar along the template the enzyme has traveled from the startpoint (see the mRNA molecules in Figure 17.24). The congregation of many polymerase molecules simultaneously transcribing a single gene increases the amount of mRNAtranscribed from it, which helps the cell make the encodedprotein in large amounts.

(~APTE~ SEVENTEEN From Gene to Protein 333

the AAVAAA signal, proteins associated with the growingRNA transcript cut it free from the polymerase, releasing thepre-mRNA. However, the polymerase continues transcribingDNA for hundreds of nucleotides past the site where the premRNA was released. Recent research on yeast cells suggeststhat the RNA produced by this continued transcription is di·gested byan enzyme that moves along the RNA. The data sup·port the idea that when the enzyme reaches the polymerase,transcription is terminated and the polymerase falls off theDNA. Meanwhile, the pre-mRNA undergoes processing, thetopic of the next section.

1. Compare DNA polymerase and RNA polymerase interms of how they function, the requirement for atemplate and primer, the direction ofsynthesis, andthe type of nucleotides used.

2, What is a promoter, and is it located at the upstreamor downstream end ofa transcription unit?

3. What makes RNA polymerase start transcribing agene at the right place on the DNA in a bacterial cell?In a eukaryotic cell?

4. -'MUI4 Suppose X-rays caused a sequencechange in the TATA box ofa particular gene's promoter. How would that affect transcription of thegene? (See Figure 17.8.)

For suggested answers, see Appendi~ A.

CONCEPT CHECI( 17.2

rior sections of the RNA molecule are cut out and the remaining parts spliced together. These modifications producean mRNA molecule ready for translation.

Alteration of mRNA Ends

Each end ofa pre-mRNA molecule is modified in a particularway (figure 17.9). The 5' end is synthesized first; it receives aS' cap, a modified form of a guanine (G) nucleotide addedonto the 5' end after transcription of the first 20 to 40 nucleotides. The 3' end of the pre-mRNA molecule is also modifiedbefore the mRNA exits the nucleus. Recall that the premRNA is released soon after the polyadenylation signal,AAUAAA, is transcribed. At the 3' end, an enzyme adds 50 to250 more adenine (A) nucleotides, forming a poly-A tail. The5' cap and poly-A tail share several important functions. First,they seem to facilitate the export of the mature mRNA fromthe nucleus. Second, they help protect the mRNA from degradation by hydrolytic enzymes. And third, they help ribosomesattach to the 5' end of the mRNA once the mRNA reaches thecytoplasm. Figure 17.9 shows a diagram of a eukaryoticmRNA molecule with cap and taiL The figure also shows theuntranslated regions (UTRs) at the 5' and 3' ends of themRNA (referred to as the 5' UTR and 3' UTR). The UTRs areparts ofthe mRNA that will not be translated into protein, butthey have other functions, such as ribosome binding.

Split Genes and RNA Splicing

r;:~:;;:i~~~~IS modify RNAafter transcription

Enzymes in the eukaryotic nucleus modifypre-mRNA in spe·cHic ways before the genetic messages are dispatched to thecytoplasm. During this RNA processing, both ends ofthe primary transcript are altered. Also, in most cases, certain inte-

Aremarkable stage of RNA processing in the eukaryotic nucleusis the removal of large portions of the RNA molecule that is initially synthesized-a cut-and-paste job called RNA splicing,similar to editing a video (Figure 17.10). The average length ofa transcription unit along a human DNA molecule is about27,000 base pairs, so the primary RNA transcript is also that long.However, it takes only 1,200 nucleotides in RNA to code for theaverage-sized protein of 400 amino acids. (Remember, eachamino acid is encoded by a triplet of nucleotides.) This meansthat most eukaryotic genes and their RNA transcripts havelong noncoding stretches of nucleotides, regions that are not

Amodified guanine nucleotideadded to the 5' end

G

~5'Cap 5'UTR

50 to 250 adenine nucleotidesadded to the 3' end

Protein-coding segment•

•3'UTR

.... Figure 17.9 RNA processing: additionof the 5' cap and poly.A tail. Enzymesmodify the two ends 0/ a eukaryotic pre-mRNAmolecule, The modi/ied ends may promote the


expon of mRNA from the nucleus. and they helpprotect the mRNA from degradation, When themRNA reaches the cytoplasm, the modified ends,in conjunction with certain cytoplasmic proteins,

facilitate ribosome attachment. The 5' cap andpoly·A tail are nol translated inlo protein. nor arethe regions called the 5' untranslated region(5' UTR) and 3' untranslaled region (3' UTR).

Pre-mRNA

mRNA

30 31

15' UTR

Exon Intron Exon 3'"Po~I,--A'-=~~" '

104 105 146

Iintrons cut out and+exons spliced together

,Poly-A tail

... Figure 17.10 RNA processing: RNAsplicing. The RNA molecule shown here codesfor l3-globin, one of the polypeptides ofhemoglobin, The numbers under the RNA referto codons; 13-globin is 146 amino acids long, The

l3-globin gene and its pre-mRNA tranSCript havethree exons, corresponding to sequences that willleave the nucleus as mRNA. (The 5' UTR and 3'UTR are parts of exons because they are includedin the mRNA; however, they do not code for

protein.) During RNA processing, the introns arecut out and the exons spliced together. In manygenes, the introns are much larger relative to theexons than they are in the l3-globin gene. (Thepre-mRNA is not drawn to scale.)

Spliceosome

RNA transcript (pre-mRNA)5',,"' """'..- _

bon 1 Intron Exon 2

SpllCeDsome Jl~components

Cut-outIntron

mRNA0 5'1

Exon 1 Exon 2

o Protein

;eRNA ~l=!IJ~ (snRNPs \

... Figure 17.11 The roles of snRNPs and spliceosomes inpre·mRNA splicing. The diagram shows only a portion of the premRNA transcript; additional introns and exons lie downstream fromthe ones pictured here, 0 Small nuclear ribonucleoproteins (snRNPs)and other proteins form a molecular complex called aspliceosome ona pre-mRNA molecule containing exons and introns. 6 Within thespliceosome, snRNA base-pairs with nucleotides at speCific sites alongthe intron. 0 The spliceosome cuts the pre-mRNA, releasing theintron, and at the same time splices the exons together. Thespliceosome then comes apart, releasing mRNA, which now containsonlyexons,

translated. Even more surprising is that most ofthese noncoding sequences are interspersed between coding segments of

the gene and thus between coding segments ofthe pre-mRNA.In other words, the sequence of DNA nucleotides that codesfor a eukaryotic polypeptide is usually not continuous; it is splitinto segments. The noncoding segments ofnucleic acid that liebetween coding regions are called intervening sequences, orintrons. The other regions are called exom, because they areeventually expressed, usually by being translated into aminoacid sequences. (Exceptions include the VTRs of the exons atthe ends of the RNA, which make up part ofthe mRNA but arenot translated into protein. Because of these exceptions, youmay find it helpful to think of exons as sequences of RNA thatexit the nucleus.) The terms intron and exon are used for both

RNA sequences and the DNA sequences that encode them.In making a primary transcript from agene, RNA polymerase

II transcribes both introns and exons from the DNA, but the

mRNA molecule that enters the cytoplasm is an abridged version. The introns are cut out from the molecule and the exonsjoined together, forming an mRNA molecule with a continuouscoding sequence. This is the process of RNA splicing.

How is pre-mRNA splicing carried out? Researchers havelearned that the signal for RNA splicing is a short nucleotidesequence at each end of an intron. Particles called small nudear ribonucleoproteins, abbreviated snRNPs (pronounced~snurps"), recognize these splice sites. As the name implies,snRNPs are located in the cell nucleus and are composed of

RNA and protein molecules. The RNA in a snRNP particle iscalled a small nuclear RNA (snRNA); each molecule is about

150 nucleotides long. Several different snRNPs join with additional proteins to form an even larger assembly called aspliceosome, which is almost as big as a ribosome. Thespliceosome interacts with certain sites along an intron, releasing the intron and joining together the two exons thatflanked the intron (Figure 17.11). There is strong evidencethat snRNAs catalyze these processes, as well as participatingin spliceosome assembly and splice site recognition.


... Figure 17.12 Correspondence between exons andprotein domains.

Exon 3 ~

17.3

Gene,

Polypeptide

Transcription !RNA processing !

Exon 1 Intron hon 2 IntronDNA

CONCEPT CHECK

Proteins often have a modular architecture consisting ofdiscrete structural and functional regions called domains.One domain of an enzymatic protein, for instance, might in~

c1ude the active site, while another might attach the protein toa cellular membrane. In quite a few cases, different exons codefor the different domains ofa protein (Figure 11.12).

The presence of introns in a gene may facilitate the evolution of new and potentially useful proteins as a result of aprocess known as exon shuffiing.lntrons increase the probability of potentially beneficial crossing over between the exons ofalleles-simply by providing more terrain for crossovers without interrupting coding sequences. We can also imagine theoccasional mixing and matching ofexons between completelydifferent (nonallelic) genes. Exon shuffling ofeither sort couldlead to new proteins with novel combinations of functions.While most of the shuffling would result in nonbeneficialchanges, occasionally a beneficial variant might arise.

1. How does alteration of the 5' and 3' ends of premRNA affect the mRNA that exits the nucleus?

2. How is RNA splicing similar to editing a video?3. A'm'n i• In nematode worms, a gene that codes

for an ATPase has two alternatives for exon 4 andthree alternatives for exon 7. How many differentforms of the protein couid be made from this gene?

For suggested answers, see Appendix A.

The Functional and EvolutionaryImportance of Introns

What could be the biological functions of introns and RNAsplicing? While specific functions may not have been identified for most introns, at least some contain sequences thatregulate gene activity. And the splicing process itself isnecessary for the passage of mRNA from the nucleus to thecytoplasm.

One consequence of the presence ofintrons in genes is thata single gene can encode more than one kind of polypeptide.Many genes are known to give rise to two or more differentpolypeptides, depending on which segments are treated as ex·ons during RNA processing; this is called alternative RNAsplicing (see Figure 18.11). For example, sex differences in fruitflies are largely due to differences in how males and femalessplice the RNA transcribed from certain genes. Results fromthe Human Genome Project (discussed in Chapter 21) suggestthat alternative RNA splicing is one reason humans can getalong with a relatively small number ofgenes-about one anda half times as many as a fruit fly. Because ofalternative splicing, the number of different protein products an organismproduces can be much greater than its number ofgenes.

Ribozymes

The idea ofa catalytic role for snRNA arose from the discovery of ribozymes, RNA molecules that function as enzymes.In some organisms, RNA splicing can occur without proteinsor even additional RNA molecules: The intron RNA functionsas a ribozyme and catalyzes its own excision! For example, inthe ciliate protist Tetrahymena, self.splicing occurs in the pro~

duction of ribosomal RNA (rRNA), a component of the or~

ganism's ribosomes. The pre-rRNA actually removes its ownintrons. The discovery of ribozymes rendered obsolete theidea that all biological catalysts are proteins.

Three properties of RNA enable some RNA molecules tofunction as enzymes. First, because RNA is single-stranded, aregion ofan RNA molecule may base-pair with a complementary region elsewhere in the same molecule, which gives themolecule a particular three-dimensional structure. A specificstructure is essential to the catalytic function of ribozymes,just as it is for enzymatic proteins. Second, like certain aminoacids in an enzymatic protein, some of the bases in RNA contain functional groups that may participate in catalysis. Third,the ability of RNA to hydrogen~bond with other nucleic acidmolecules (either RNA or DNA) adds spe<ifkity to its catalyticactivity. For example, complementary base pairing bern'eenthe RNA of the spliceosome and the RNA of a primary RNAtranscript precisely locates the region where the ribozyme cat·alyzes splicing. Later in this chapter, you will see how theseproperties of RNA also allow it to perform important noncatalytic roles in the cell, such as recognition of the threenucleotide codons on mRNA.


The Structure and Function of Transfer RNA

... Figure 17.13 Translation: the basic concept. As a moleculeof mRNA is moved through a ribosome. codons are translated intoamino acids, one by one, The interpreters are tRNA molecules. eachtype with a specific anticodon at one end and acorresponding aminoacid at the other end. A tRNA adds its amino acid cargo to agrowingpolypeptide chain when the anticodon hydrogen-bonds to acomplementary codon on the mRNA. The figures that follow showsome of the details of translation in a bacterial cell.

••Aminoacids

~

••

••

•

••

mRNA

•• Polypeptide

BiofLix Visit the Study Areaat www.masteringbio.comfor the BioFlix 3-0 Animation onProtein SynthesiS

•

Like mRNA and other types ofcellular RNA, transfer RNA mol

ecules are transcribed from DNA templates. In a eukaryotic cell,

tRNA, like mRNA, is made in the nucleus and must travel from

the nucleus to the cytoplasm, where translation occurs. In both

bacterial and eukaryotic cells, each tRNA molecule is used re

peatedly, picking up its designated amino acid in the cytosol, de

positing this cargo onto a polypeptide chain atthe ribosome, and

then leaving the ribosome, ready to pick up another amino acid.

A tRNA molecule consists of a single RNA strand that is

only about 80 nucleotides long (compared to hundreds of nu

cleotides for most mRNA molecules). Because of the presence

of complementary stretches of bases that can hydrogen-bond

to each other, this single strand can fold back upon itself and

form a molecule with a three-dimensional structure. Flattened

into one plane to reveal this base pairing, a tRNA molecule

Molecular Components ofTranslation

r;~:~s~:~o~~~~he RNA-directedsynthesis of a polypeptide:a closer look

We will now examine in greater detail how genetic informa

tion flows from mRNA to protein-the process of translation.

As we did for transcription, we'll concentrate on the basic

steps oftranslation that occur in both bacteria and eukaryotes,while pointing out key differences.

In the process of translation, a cell interprets a genetic mes

sage and builds a polypeptide accordingly. The message is aseries of codons along an mRNA molecule, and the interpreter is called transfer RNA (tRNA). The function oftRNA is to transfer amino acids from the cytoplasmic pool

of amino acids to a ribosome. A cell keeps its cytoplasmstocked with all 20 amino acids, either by synthesizing them

from other compounds or by taking them up from the sur

rounding solution. The ribosome adds each amino acid

brought to it by tRNA to the growing end of a polypeptidechain (Figure 17.13).

Molecules of tRNA are not all identical. The key to

translating a genetic message into a specific amino acid se

quence is that each type oftRNA molecule translates a par

ticular mRNA codon into a particular amino acid. As a

tRNA molecule arrives at a ribosome, it bears a specific

amino acid at one end. At the other end of the tRNA is a nu

cleotide triplet called an anticodon, which base-pairs with

a complementary codon on mRNA. For example, considerthe mRNA codon UUU, which is translated as the amino

acid phenylalanine. The tRNA that base-pairs with this

codon by hydrogen bonding has AAA as its anticodon and

carries phenylalanine at its other end (see the middle tRNA

on the ribosome in Figure 17.13). As an mRNA molecule is

moved through a ribosome, phenylalanine will be added to

the polypeptide chain whenever the codon UUU is pre

sented for translation. Codon by codon, the genetic mes

sage is translated as tRNAs deposit amino acids in the order

prescribed, and the ribosome joins the amino acids into a

chain. The tRNA molecule is a translator because it can

read a nucleic acid word (the mRNA codon) and interpret

it as a protein word (the amino acid).

Translation is simple in principle but complex in its bio

chemistry and mechanics, especially in the eukaryotic cell. In

dissecting translation, we'll concentrate on the slightly less

complicated version of the process that occurs in bacteria.

Let's first look at the major components in this cellular

process. Then we will see how they act together to make a

polypeptide.


3'

Amino acidattachment site

, 5'C 'GG • CC • GC GC .,, • C

Aminoacyl-tRNAsynthetase (enzyme)

oActive site bindsthe amino acid andATP.

AminO<lcyl-tRNAsynthetase

I

Computer model

eATP loses two® groupsand joins amino acid as AMP.

OThe tRNA chargedf\r-----iB---\-iwith amino acid is

released by the enzyme.

tRNA

iEXEJ,I

~, ~,

... Figure 17.15 An aminoacyl-tANA synthetase joining aspecific amino acid to a tANA. Linkage of the tRNA and amino acidIS an endergonic process that occurs at the expense of ATP The ATP loses\IAIo phosphate groups, becoming AMP (adenOSine monophosphate)

end, which is the attachment site for an amino acid. Thus, thestructure of a tRNA molecule fits its function.

The accurate translation of a genetic message requires twoprocesses that involve molecular recognition. Rrst, a tRNA thatbinds to an mRNA codon specifying a particular amino acidmust carry that amino acid, and no other, to the ribosome. Thecorrect matching up of tRNA and amino acid is carried out by afamily of related enzymes called antinoacyl-tRNA synthetases(Figure 17.15). The active site of each type of aminoacyl-tRNA

oAppropriatetRNA covalentlybonds to amino f----c-\acid, displacing

AMP.

®-J Adermine IAMP

Amino acidattachment site

"'"'"

(c) Symbol usedin this book

3'

Hydrogenbonds

(b) Three-dimensional structure

(a) Two-dimensional structure. The four base-paired regions andthree loops are charaderistic of all tRNAs, as is the basesequence of the amino acid attachment site allhe 3' end. Theanticodon triplet is unique to each IRNA type, as are somesequences in the other two loops. (The asterisks mark basesthat have been chemically modified, a characteristic of tRNA.)

... Figure 17.14 The structure of transfer RNA (tRNA).Anticodon, afe conventionally written]' • S' to align properly withcodons written 5' .]' (see Figure 17,13), For base pairing, RNAstrands must be anliparallel, like DNA. For example, anticodon3'-AAG-5' pairs with mRNA codon 5'-UU(-3'

C A ( A

looks like a cloverleaf (Figure 17.14a). TIle tRNA actuallytv.'ists and folds into a compact three-dimensional structurethat is roughly L-shaped (Figure 17.14b). The loop extendingfrom one end of the L includes the anticodon, the particularbase triplet that base-pairs to a specific mRNA codon. Fromthe other end of the L-shaped tRNA molecule protrudes its 3'


... Figure 17.16 The anatomy of a functioning ribosome.

(b) Schematic model showing binding sites. A ribosome has anmRNA binding site and three tRNA binding sites, known as theA, P, and Esites. This schematic ribosome will appear in laterdiagrams.

Smallsubunit

LargesubUnit

tRNA

"

Next amino acidto be added topolypeptide chain

A site (~minoacyl

tRNA binding site)

Growing polypeptideAmino end----'",

Esite(yit site)

mRNA---Ibinding site

mRNA

tRNAmolecules

(c) Schematic model with mRNA and tRNA. A tRNA fits into abinding site when its anticodon base-pairs with an mRNAcodon, The Psite holds the tRNA attached to the growingpolypeptide. The A site holds the tRNA carrying the next aminOacid to be added to the polypeptide chain. Discharged tRNAleaves from the Esite.

(a) Computer model of functioning ribosome. This is a model ofa bacterial ribosome, showing its overall shape, The eukaryoticribosome is roughly similar A ribosomal subunit is an aggregateof ribosomal RNA molecules and proteins.

Psite (feptidyl-tRNAbinding site)

Ribosomes

Ribosomes facilitate the specific coupling oftRNA anticodonswith mRNA codons during protein synthesis. A ribosome ismade up of two subunits, called the large and small subunits(Figure 17.16). The ribosomal subunits are constructed ofproteins and RNA molecules named ribosomal RNAs, orrRNAs. In eukaryotes, the subunits are made in the nucleolus.Ribosomal RNA genes on the chromosomal DNA are transcribed, and the RNA is processed and assembled with proteins imported from the cytoplasm. The resulting ribosomalsubunits are then exported via nuclear pores to the cytoplasm.In both bacteria and eukaryotes, large and small subunits jointo form a functional ribosome only when they attach to anmRNA molecule. About two-thirds ofthe mass ofa ribosomeconsists of rRNAs, either three molecules (in bacteria) or four(in eukaryotes). Because most cells contain thousands of ribosomes, rRNA is the most abundant type ofcellular RNA.

Although the ribosomes of bacteria and eukaryotes arevery similar in structure and function, those ofeukaryotes areslightly larger and differ somewhat from bacterial ribosomesin their molecular composition. The differences are medicallysignificant. Certain antibiotic drugs can inactivate bacterial ribosomes without inhibiting the ability of eukaryotic ribosomes to make proteins. These drugs, including tetracyclineand streptomycin, are used to combat bacterial infections.

synthetase fits only a specific combination of amino acid andtRNA. There are 20 different synthetases, one for each aminoacid; each synthetase is able to bind all the different tRNAs thatcode for its particular amino acid. The synthetase catalyzes thecovalent attachment of the amino acid to its tRNA in a processdriven by the hydrolysis of ATP. The resulting aminoacyl tRNA,also called a charged tRNA, is released from the enzyme and isthen available to deliver its amino acid to a growing polypeptidechain on a ribosome.

The second recognition process involves matching up thetRNA anticodon with the appropriate mRNA codon. If onetRNA variety existed for each mRNA codon that specifies anamino acid, there would be 61 tRNAs (see Figure 17.5). In fact,there are only about 45, signifying that some tRNAs must be ableto bind to more than one codon. Such versatility is possible because the rules for base pairing between the third base ofa codonand the corresponding base of a tRNA anticodon are relaxedcompared to those at other codon positions. For example, thebase U at the 5' end ofa tRNA anticodon can pair with either Aor G in the third position (at the 3' end) ofan mRNA codon. Theflexible base pairing at this codon position is called wobble.Wobble explains why the synonymous codons for agiven aminoacid can differ in their third base, but usually not in their otherbases. For example, a tRNA with the anticodon 3'-UCU-5' canbase-pair with either the mRNA codon 5'-AGA-3' or 5'-AGG3', both ofwhich code for arginine (see Figure 17.5).


The structure of a ribosome reflects its function of bringing mRNA together with tRNAs carrying amino acids. In addition to a binding site for mRNA, each ribosome has threebinding sites for tRNA (see Figure 17.16). The P site (peptidyl

tRNA site) holds the tRNA carrying the growing polypeptidechain, while the A site (aminoacyl-tRNA site) holds thetRNA

carrying the next amino acid to be added to the chain. Discharged tRNAs leave the ribosome from the E site (exit site).

The ribosome holds the tRNA and mRNA in close proximityand positions the new amino acid for addition to the carboxylend of the growing polypeptide. It then catalyzes the formation of the peptide bond. As the polypeptide becomes longer,it passes through an exit tUn/lei in the ribosome's large subunit. When the polypeptide is complete, it is released to thecytosol through the exit tunnel.

Recent research strongly supports the hypothesis thatrRNA, not protein, is primarily responsible for both the structure and the function ofthe ribosome. The proteins, which are

largely on the exterior, support the shape changes ofthe rRNAmolecules as they carry out catalysis during translation. Ribosomal RNA is the main constituent of the interface betweenthe tv.'o subunits and of the A and P sites, and it is the catalystof peptide bond formation. Thus, a ribosome can be regardedas one colossal ribozyme!

Building a Polypeptide

We can divide translation, the synthesis of a polypeptidechain, into three stages (analogous to those of transcription);

initiation, elongation, and termination. All three stages require protein "factors" that aid in the translation process. Forcertain aspects of chain initiation and elongation, energy is

also required. It is provided by the hydrolysis of GTP (guanosine triphosphate), a molecule closely related to ATP.

Ribosome Association and Initiation ofTranslation

The initiation stage of translation brings together mRNA, atRNA bearing the first amino acid of the polypeptide, and thetwo subunits of a ribosome (Figure 17.17). First, a small ribosomal subunit binds to both mRNA and a specific initiatortRNA, which carries the amino acid methionine. In bacteria,the small subunit can bind these two in either order; it bindsthe mRNA at a specific RNA sequence, just upstream of thestart codon, AUG. In eukaryotes, the small subunit, with theinitiatortRNA already bound, binds to the 5' cap ofthe mRNAand then moves, or scans, downstream along the mRNA until

it reaches the start codon, and the initiator tRNA hydrogenbonds to it. In either case, the start codon signals the start oftranslation; this is important because it establishes the codonreading frame for the mRNA.

The union of mRNA, initiator tRNA, and a small riboso

mal subunit is followed by the attachment of a large ribosomal subunit, completing the translation initiation complex.Proteins called initiation factors are required to bring allthese components together. The cell also expends energy inthe form of a GTP molecule to form the initiation complex.

At the completion of the initiation process, the initiatortRNA sits in the P site of the ribosome, and the vacant A site

is ready for the next aminoacyl tRNA. Note that a polypeptide is always synthesized in one direction, from the initialmethionine at the amino end, also caned the N-terminus, to

ward the final amino acid at the carboxyl end, also called theC-terminus (see Figure 5.18).

3'

largeribosomalsubunit

Translation initiation complex

f) The arrival of a large ribosomal subunitcompletes the initiallon complex,Proteins called initiation factors (notshown) are required to bring all thetranslation components together, GTPprovides the energy for the assembly.The initiator tRNA is in the Psite; the Asite is available to the tRNA bearing thenext aminO acid

$ GDP

g"3'

Q~

mRNA binding site

Smallribosomalsubunit

oA small ribosomal subUnit binds to amolecule of mRNA. In a bacterial cell, themRNA binding site on this subunitrecognizes a specific nucleotide sequenceon the mRNA just upstream of the startcodon. An initiator tRNA, with theanticodon UAC, base·palrs with the startcodon. AUG This tRNA carries the aminOacid methionine (Met)

"

Initiator tRNA--+-

... Figure 17.17 The initiationof translation.


t)Peptide bond formation.An rRNA molecule of thelarge ribosomal subunitcatalyzes the formation of apeptide bond between thenew amino acid in the A siteand the carboxyl end of thegrowing polypeptide inthe Psite. This step removesthe polypeptide from thetRNA in the P site andattaches it to the amino acidon the tRNA in the A site.

l1GCodon recognition. The anticodonof an incoming aminoacyl tRNA basepairs with the complementary mRNA

+: codon in the A site. Hydrolysis of GTPincreases the accuracy and efficiencyof this step.

3'

m~

5'

Amino endof polypeptide

oTranslocation. Theribosome translocates thetRNA in the A site to theP site. At the same time,the empty tRNA in the Psite is moved to the Esite,where it is released. ThemRNA moves along with itsbound tRNAs, bringing thenext codon to be translatedinto the A site.

Ribosome ready for ~ /next amlOoacyl tRNj

~

I "''''''''''''ION I I ()N,I,

~I M~I TW§4!T!I!i )E

... Figure 17.18 The elongation cycle of translation. The hydrolysis of GTP plays animportant role in the elongation process. Not shown are the proteins called elongation factors.

Elongation of the Polypeptide Chain Termination of Translation

In the elongation stage oftranslation, amino acids are added oneby one to the pnxeding amino acid. Each addition involves theparticipation of several proteins called elongation factors andoccurs in a three-step cycle described in Figure 17.18. Energyexpenditure occurs in the first and third steps. Codon recognition requires hydrolysis of one molecule of GTP, which increases the accuracy and efficiency ofthis step. One more GTPis hydrolyzed to provide energy for the translocation step.

The mRNA is moved through the ribosome in one directiononly,S' end first; this is equivalent to the ribosome moving5' ---+ 3' on the mRNA. The important point is that the ribosome and the mRNA move relative to each other, unidire<tionally, codon by codon. The elongation cycle takes less thana tenth ofa second in bacteria and is repeated as each amino acidis added to the chain until the polypeptide is completed.

The final stage of translation is termination (Figure 17.19, onthe next page). Elongation continues until a stop codon in themRNA reaches the A site of the ribosome. The base tripletsVAG, VAA, and VGA do not code for amino acids but insteadact as signals to stop translation. A protein called a releasefactor binds directly to the stop codon in the A site. The releasefactor causes the addition of a water molecule instead of anamino acid to the polypeptide chain. This reaction breaks (hydrolyzes) the bond between the completed polypeptide andthe tRNA in the P site, releasing the polypeptide through theexit tunnel ofthe ribosome's large subunit (see Figure 17.l6a).The remainder of the translation assembly then comes apartin a multistep process, aided by other protein factors. Break·down of the translation assembly requires the hydrolysis oftwo more GTP mole<ules.


€) The two ribosomal subunits and theother components of the assemblydissociate.

"

Stop codon(UAG, UAA, or UGA)

o When a ribosome reaches astop codon onmRNA, the A site althe ribosome accepts a

release factor," a protein shaped like atRNA, instead of an aminoacyl tRNA.

t) The release factor promotes hydrolysis of thebond between the tRNA in the Psite and thelast amino acid of the polypeptide, thusfreeing the polypeptide from the ribosome.

- "

.... Figure 17.19 The termination of translation. like elongation, termination reqUires GTPhydrolysis as well as additional protein fadors, which are not shown here.

Start ofmRNA(S' end)

Polyribosomes

A single ribosome can make an average-sized polypeptide inless than a minute. Typically, however, multiple ribosomestranslate an mRNA at the same time; that is, a single mRNA isused to make many copies of a polypeptide simultaneously.Once a ribosome moves past the start codon, a second ribosome can attach to the mRNA, eventually resulting in a number of ribosomes trailing along the mRNA. Such strings ofribosomes, called polyribosomes (or polysomes), can be seenwith an electron microscope (figure 17.20). Polyribosomesare found in both bacterial and eukaryotic cells. They enable acell to make many copies ofa polypeptide very quickly.

Growingpolypeptides

Incomin~ribosomalsubunits/

End ofmRNA(3' end)

(a) An mRNA molecule is generally translated simultaneouslyby several ribosomes In clusters called polyribosomes.

in the cell. Certain amino acids may be chemically modified bythe attachment ofsugars, lipids, phosphate groups, or other additions. Enzymes may remove one or more amino acids fromthe leading (amino) end of the polypeptide chain. In somecases, a polypeptide chain may be enzymatically cleaved into

Completing and Targetingthe Functional Protein

The process of translation is often not sufficient to make afunctional protein. In this section, you wi11learn about modifications that polypeptide chains undergo after the translationprocess as well as some ofthe mechanisms used to targetcom~

pleted proteins to specific sites in the cell.

Protein Folding and Post-Translational Modifications

During its synthesis, a polypeptide chain begins to coil andfold spontaneously as a consequence of its amino acid sequence (primary structure), forming a protein with a specificshape: a three-dimensional molecule with secondary and ter~

tiary structure (see Figure 5.21). Thus, a gene determines pri~

mary structure, and primary structure in turn determinesshape. In many cases, a chaperone protein (chaperonin) helpsthe polypeptide fold correctly (see Figure 5.24).

Additional steps-post·transiLltional modifications-maybe required before the protein can begin doing its particular job


(b) This micrograph shows a large polyribosomein a bacterial cell (TEM),

... Figure 17.20 Polyribosomes.

a,1l1m

two or more pieces. For example, the protein insulin is first synthesized as a single polypeptide chain but becomes active onlyafter an enzyme cuts out a central part of the chain, leaving aprotein made up oftwo polypeptide chains connected by disulfide bridges. In other cases, w..-o or more polypeptides that aresynthesized separately may come together, becoming the subunits ofa protein that has quaternary structure. A familiar example is hemoglobin (see Figure 5.21).

Targeting Polypeptides to Specific l.ocations

In electron micrographs of eukaryotic cells active in proteinsynthesis, hvo populations of ribosomes (and polyribosomes)are evident: free and bound (see Figure 6.11). Free ribosomesare suspended in the cytosol and mostly synthesize proteinsthat stay in the cytosol and function there. In contrast, boundribosomes are attached to the cytosolic side of the endoplasmic reticulum (ER) or to the nuclear envelope. Bound ribosomes make proteins of the endomembrane system (thenuclear envelope, ER, Golgi apparatus, Iysosomes, vacuoles,and plasma membrane) as well as proteins secreted from thecell, such as insulin. The ribosomes themselves are identicaland can switch their status from free to bound.

\X'hat determines whether a ribosome will be free in the cytosol or bound to rough ER at any given time? Polypeptide synthesis always begins in the cytosol, when a free ribosome starts totranslate an mRNA molecule. There the process continues tocompletion-unless the growing polypeptide itself cues the ri·bosome to attach to the ER. The polypeptides of proteins destined for the endomembrane system or for secretion aremarked by asignal peptide, which targets the protein to the ER(Figure 17.21). The signal peptide, a sequence of about 20amino acids at or near the leading (amino) end of the polypeptide, is recognized as it emerges from the ribosome by a proteinRNA complex called a signal-recognition particle (SRP).This particle functions as an adapter that brings the ribosometo a receptor protein built into the ER membrane. This receptoris part of a multiprotein translocation complex. Polypeptidesynthesis continues there, and the growing polypeptide snakesacross the membrane into the ER lumen via a protein pore. Thesignal peptide is usually removed by an enzyme. The rest of thecompleted polypeptide, ifit is to be secreted from the cell, is released into solution within the ER lumen (as in Figure 17.21). Alternatively, if the polypeptide is to be a membrane protein, itremains partially embedded in the ER membrane.

o Polypeptidesynthesis beginson a freeribosome inthe cytosol.

f)An SRP binds tothe signal peptide.halting synthesismomentarily.

f)The SRP binds to areceptor protein in the ERmembrane. This receptoris part of a protein complex(a translocation complex)that has a membrane poreand a signai-cleaving enzyme.

oThe SRP leaves, andpolypeptide synthesisresumes, with simultaneous translocation acrossthe membrane. (Thesignal peptide staysattached to the translocation complex.)

0The signalcleaving enzymecuts off thesignal peptide.

0The rest ofthe completedpolypeptide leavesthe ribosome andfolds into its finalconformation.

Signalrecognitionparticle(SRP)

SRPreceptor (

CYTOSOL protein~

=--------------ER LUMEN

... Figure 17.21 The signal mechanismfor targeting proteins to the Eft. Apolypeptide destined for the endomembranesystem or for secretion from the cell begins

with a signal peptide, a series of amino acidsthat targets it for the ER, This figure shows thesynthesis of asecretory protein and itssimultaneous import into the ER, In the ER and

then in the Golgi, the protein will be processedfurther, Finally, a transport vesicle will convey itto the plasma membrane for release from thecell (see Figure 7.10).


• Figure 17.22 The molecular basis of sickle-cell disease: a point mutation. The allelethat causes sickle-cell disease differs from the wild-type (normal) allele by a slngle DNA base pair.

mRNA mRNA The mutant mRNA5' i:==EEIEC==]3' 5'===](~_1!1.1I;11===:3' has aUinstead ofan Ain one codon.

Wild-type hemoglobin DNA Mutant hemoglobin DNA

"~~~~!~.:.:'~~~~5' 3,·~~~~t«:;:.:'~~~~5'5'= tcaWZJ 3' 5': N-a 3'

Normal hemoglobin

~

The mutant (sICkle·cell) hemoglobin hasa ~ahne (Val) insteadof a glutamic acid(Glu).

In the DNA, themutant templatestrand (top) has anAwhere the wildtype template hasor.

Sickle·cell hemoglobin

~

Point mutations within a gene can be divided into two general categories: base-pair substitutions and base-pair insertions or deletions. Let's now consider how these mutationsaffect proteins.

Types of Point Mutations

Substitutions

A base-pair substitution is the replacement of one nucleotide and its partner with another pair of nucleotides(Figure 17.23a). Some substitutions are called silent

mutations because, owing to the redundancy of the geneticcode, they have no effect on the encoded protein. In otherwords, a change in a base pair may transform one codon intoanother that is translated into the same amino acid. For example, if 3'-CCG-5' on the template strand mutated to3'-CCA-5', the mRNA codon that used to be GGC wouldbecome GGU, but a glycine would still be inserted at theproper location in the protein (see Figure 17.5). Substitutions that change one amino acid to another one are calledmissense mutations. Such a mutation may have little effecton the protein: The new amino acid may have propertiessimilar to those of the amino acid it replaces, or it may be ina region of the protein where the exact sequence of aminoacids is not essential to the protein's function.

production of an abnormal protein (figure 17.22; also seeFigure 5.22). In individuals who are homozygous for the mutant allele, the sickling of red blood cells caused by the alteredhemoglobin produces the multiple symptoms associated withsickle-cell disease (see Chapter 14). Another example is aheart condition responsible for some incidents of suddendeath in young athletes, called familial cardiomyopathy. Pointmutations in several genes have been identified, each ofwhich can lead to this disorder.

17.4CONCEPT CHECI(

1. What two processes ensure that the correct aminoacid is added to a growing polypeptide chain?

2. Describe how the formation of polyribosomes canbenefit the celL

3. Describe how a polypeptide to be secreted is transported to the endomembrane system.

4. -'M"'I. Discuss the ways in which rRNA structure likely contributes to ribosomal function.

For suggested answers. see Appendix A

r;::~t'~:~~~~s can affectprotein structure and function

Now that you have explored the process ofgene expression, youare ready to understand the effects ofchanges to the genetic information ofa cell (or virus). These changes, called mutations,are responsible for the huge diversity ofgenes found among organisms because mutations are the ultimate source of newgenes. In Rgure 15.15, we considered large-scale mutations,chromosomal rearrangements that affectlong segments ofDNA. Here we examinepoint mutations, chemical changes in asingle base pair of a gene.

Ifa point mutation occurs in a gameteor in a cell that gives rise to gametes, itmay be transmitted to offspring and to asuccession of future generations. If themutation has an adverse effect on thephenotype of an organism, the mutantcondition is referred to as a genetic disorder or hereditary disease. For example,we can trace the genetic basis of sicklecell disease to the mutation of a singlebase pair in the gene that encodes thej3-globin polypeptide of hemoglobin.The change of a single nucleotide in theDNA's template strand leads to the

Other kinds of signal peptides are used to target polypeptides to mitochondria, chloroplasts, the interior of the nucleus,and other organelles that are not part of the endomembranesystem. The critical difference in these cases is that translationis completed in the cytosol before the polypeptide is importedinto the organelle. The mechanisms of translocation also vary,but in all cases studied to date, the "zip codes~ that address proteins for secretion or to cellular locations are signal peptides ofsome sort. Bacteria also employ signal peptides to target proteins for secretion.


Wild type

DNA template strand 3'S'

TACTTCAAACCGATT

A T G A A G T T T G G C T A A

S'

3'

mRNA S'

Protein

Amino end

AUG A A G U U U G G C U A A 3'

A instead of G Extra A

3'S'

TACTTCAAACCAATT

ATGAAGTTTGGTTAA

S'3'

3'S'

TACATTCAAACCGATT

ATGTAAGTTTGGCTAA

S'

3'

U instead of C Extra U

3'AUGUAAGUUUGGCUAAs'

-¥3'

'---v---'Stop

AUGAAGUUUGGUUAAS'

Silent (no effect on amino acid sequence) Frameshilt causmg immediate nonsense (1 base-pair insertion)

T instead of ( rJ missing

A T G A A G T T G G C T A A

3'

S'

T ACT T C A A A T C GAT T

A T G A A G T T TAG C T A A

S'3'

3'

S'

TACTTCAA CCGATT S'3'

A instead of G mmissing

",3'AUGAAGUUGGCUAAS'3''---v---'

Stop

AUGAAGUUUAGCUAAS'

Missense Frameshift causing extensive missense (1 base-pair deletion)

A instead of T ~missing

3'S'

TACATCAAACCGATT

ATGTAGTTTGGCTAA

S'3'

3'S'

TACAAACCGATT

ATGTTTGGCTAA

S'3'

U instead of A

S'''I.''~.'''I.I.I'''I.I.'.''3'-¥

Nonsense

~missing

S' 3'

----~~SIOp

No frameshift, but one amino acid missing (3 base-pair deletion),A 3 base-pair insertion (not shown) would lead to an extra amino acid,

(a) Base-pair substitution (b) Base-pair insertion or deletion

... Figure 17.23 Types of point mutations, Mutations are changes in DNA that result in changes in the mRNA,

However, the base-pair substitutions ofgreatest interest arethose that cause a major change in a protein. The alteration of

a single amino acid in a crucial area ofa protein-such as in thepart of hemoglobin shown in Figure 17.22 or in the active siteof an enzyme-will significantly alter protein activity. Occasionally. such a mutation leads to an improved protein or onewith novel capabilities, but much more often such mutationsare detrimental, leading to a useless or less active protein thatimpairs cellular function.

Substitution mutations are usually missense mutations;that is, the altered codon still codes for an amino acid andthus makes sense, although not necessarily the right sense.

But a point mutation can also change a codon for an aminoacid into a stop codon. This is called a nonsense mutation,

and it causes translation to be terminated prematurely; theresulting polypeptide will be shorter than the polypeptideencoded by the normal gene. Nearly all nonsense mutationslead to nonfunctional proteins.

Insertions and Deletions

Insertions and deletions are additions or losses of nucleotidepairs in a gene (Figure 17.23b). These mutations have a disastrouseffecton the resulting protein more often than substitutions


1. What happens when one nucleotide pair is lost fromthe middle of the coding sequence ofa gene?

2. -QJ:t.illg A gene whose template strand containsthe sequence 3'·TACTTGTCCGATATe-S' is mu·tated to 3'-TACTTGTCCAATATe-S'. For both normal and mutant genes, draw the double-strandedDNA, the resulting mRNA, and the amino acid sequence each encodes. \Vhat is the effect of the mutation on the amino acid sequence?

For suggested answers. see Appendix A.

do. Insertion or deletion of nucleotides may alter the readingframe of the genetic message, the triplet grouping of bases onthe mRNA that is read during translation. Such a mutation,called a framcshift mutation, wiII occur whenever the numberof nucleotides inserted or deleted is not a multiple of three. Allthe nucleotides that are downstream ofthe deletion or insertionwill be improperly grouped into codons, and the result will beextensive missense, usually ending sooner or later in nonsenseand premature termination. Unless the frameshift is very nearthe end ofthe gene, the protein is almost certain to be nonfunctional.

CONCEPT CHECK 17.5

Mutagens

Mutations can arise in a number ofways. Errors during DNAreplication or recombination can lead to base-pair substitutions, insertions, or deletions, as well as to mutations affecting longer stretches of DNA. If an incorrect base is added toa growing chain during replication, for example, that basewill then be mismatched with the base on the other strand.In many cases, the error will be corrected by systems youlearned about in Chapter 16. Otherwise, the incorrect basewill be used as a template in the next round of replication, resulting in a mutation. Such mutations are called spontaneous

mutations. It is difficult to calculate the rate at which suchmutations occur. Rough estimates have been made of therate of mutation during DNA replication for both E. coli andeukaryotes, and the numbers are similar: About one nucleotide in every 1010 is altered, and the change is passed onto the next generation of cells.

Anumber ofphysical and chemical agents, called mutagens,interact with DNA in ways that cause mutations. In the 1920s,Hermann Muller discovered that X-rays caused geneticchanges in fruit flies, and he used X-rays to make Drosophila

mutants for his genetic studies. But he also recognized analarming implication of his discovery: X-rays and other formsof high-energy radiation pose hazards to the genetic materialof people as well as laboratory organisms. Mutagenic radiation, a physical mutagen, includes ultraviolet (UV) light,which can cause disruptive thymine dimers in DNA (seeFigure 16.18).

Chemical mutagens fall into several categories. Baseanalogs are chemicals that are similar to normal DNA basesbut that pair incorrectly during DNA replication. Some otherchemical mutagens interfere with correct DNA replication byinserting themselves into the DNA and distorting the doublehelix. Still other mutagens cause chemical changes in basesthat change their pairing properties.

Researchers have developed various methods to test themutagenic activity of chemicals. A major application of thesetests is the preliminary screening ofchemicals to identifythosethat may cause cancer. This approach makes sense becausemost carcinogens (cancer-causing chemicals) are mutagenic,and conversely, most mutagens are carcinogenic.

3% UNIT THREE Genetics

r:~~:J;:? ~~pression differsamong the domains of life, theconcept of a gene is universal

Although bacteria and eukaryotes carry out transcription andtranslation in very similar ways, we have noted certain differencesin cellular machinery and in details ofthe processes in these twodomains. The division of organisms into three domains was established about 40 years ago, when archaea were recognized asdistinct from bacteria. Like bacteria, archaea are prokaryotes.However, archaea share many aspects ofthe mechanisms ofgeneexpression with eukaryotes, as well as a few with bacteria.

Comparing Gene Expression in Bacteria,Archaea, and Eukarya

Recent advances in molecular biology have enabled researchersto determine the complete nucleotide sequences of hundreds ofgenomes, including many genomes from each domain. Thiswealth of data allows us to compare gene and protein sequencesacross domains. Foremost among genes of interest are those thatencode components ofsuch fundamental biological processes astranscription and translation.

Bacterial and eukaryotic RNA polymerases differ significantly from each other. In contrast, the single RNA polymerase of archaea resembles the three eukaryotic RNApolymerases, and archaea and eukaryotes use a complex set oftranscription factors, unlike bacteria. Transcription is terminated differently in bacteria and eukaryotes. Here again, thelittle that is known about archaeal transcription terminationsuggests it may be more like the eukaryotic process.

As far as translation is concerned, bacterial and eukaryoticribosomes are slightly different. Archaeal ribosomes are thesame size as bacterial ribosomes, but their sensitivity to chemical inhibitors most closely matches that of eukaryotic ribosomes. We mentioned earlier that initiation of translation isslightlydifferent in bacteria and eukaryotes. In this respect, thearchaeal process is more like that of bacteria.

... Figure 17.24 Coupled transcription and translation inbacteria. In badenal cells. the translation of mRNA can begin assoon as the leading (5') end of the mRNA molecule peels away fromthe DNA template. The micrograph ('TEM) shows a strand of E. coliDNA being transaib£od by RNA polymerase molecules. Attached 10eilch RNA polymerase molecule IS a gr(M'lng strand of mRNA, whIChis already being Iranslated by ribosomes. The new1y synthesizedpolypeptides are not VI5Ible In the mICrograph but are shown In thedIagram.n Which one of rhe mRNA molecules srarred rrarnaiprion flffi? On.. mar mRNA, which ribosome sraned rranslating first?

17.6CONCEPT CHECK

What Is a Gene! Revisiting the QuestionOur definition ofa gene has evolved over the past few chapters,as it has through the history of genetics. We began with theMendelian conceptofagene as adiscrete unit ofinheritance thataffects a phenotypic character (Chapter 14). We saw thatMorgan and his coUeagues assigned such genes to specifIC locion chromosomes (Olapter IS). We went on to yjey,. ageoe asaregion of specific nucleotide sequence along the length of aDNA molecule in a chromosome (Cbapter 16). Finally, in thischapter, we have considered a functional definition ofa gene asa DNA sequence that codes for a specific polypeptide chain.(Figure 17.25, on the next page, summarizes the path from geneto polypeptide in a eukaryotic eel!.) All these definitions are lISe

ful, depending on the context in \\nich genes are being studied.Clearly, the statement that a gene codes for a polypeptide is

too simple. Most eukaryotic genes contain noncoding segments (introns), so large portions of these genes have no corresponding segments in polypeptides. Molecular biologistsalso often include promoters and certain other regulatory regionsofDNA within the boundaries ofa gene. These DNA sequences are not transcribed, but they can be considered part ofthe functional gene because they must be present for transcription to occur. Our moleculardefinition ofagene must alsobe brood enough to include the DNA that is transcribed intorRNA, tRNA, and other RNAs that are not translated. Thesegenes have no polypeptide products but play crucial roles inthe ceU_ Thus, we arrive at the follo....':ing definition: Agene is aregion of DNA that am be expressed to prodm:e a final functional product that is eidler a po/ypeptideoran RNA molecule.

\Vhen considering phenotypes, however, it is often usefulto start by focusing on genes that code for polypeptides. In thiSchapter, you have learned in molecular terms how a typicalgene is expressed-by transcription into RNA and then translation into a polypeptide that forms a protein ofspecific structure and function. Proteins, in turn, bring about an organism'sobservable phenotype.

Agiven type ofcell expresses only asubset ofits genes. Thisis an essential feature in multicellular organisms: You'd be introuble if the lens cells in your eyes started. expreSSing thegenes for hair proteins, which are normally expressed only inhair follicle cells! Gene expression is precisely regulated. Wellexplore gene regulation in the next chapter, beginning withthe simpler case of bacteria and continuing with eukaryotes.

For suggested answers. see AppendiX A.

I. Would the coupling or processes shown. in Figure 17.24be found in a eukaryotic cell? Explain.

2. -lim ilI4 In eukaryotic cells, mRNAs have beenfound to have a circular arrangement in which thepoly-A tail is held by proteins near the 5' end cap. Howmight this increase translation efficiency?

O_lS~

DNA

Directlon oftranscnptton --~.

mRNA (5' end)

RNApolymerase

PO~flbosome{Polypeptide(amino end)

The most important differences between bacteria and eukaryotes with regard to gene expression arise from the bacterial cell's relative absence ofcompartmental organization. Likea one-room workshop, a bacterial cell ensures a streamlinedoperation. In the absence of a nucleus, it can simultaneouslytranscribe and translate the same gene (Figure 17.24), andthe newly made protein can quickly diffuse to its site of function. Little is currently known about whether the processes oftranscription and translation are coupled like this in archaealcells, but most researchers suspect that they are, since archaealack a nuclear envelope. In contrast, the eukaryotic cell's nuclear envelope segregates transcription from translation andprovides a compartment for extensive RNA processing. Thisprocessing stage includes additional steps whose regulationcan help coordinate the eukaryotic cell's elaborate activities(see Chapter 18). Finally, eukaryotic cells have complicatedmechanisms for targeting proteins to the appropriate cellularcompartment (organelle).

Learning more about the proteins and RNAs involved in archaeal transcription and translation will tell us much about theevolution of these processes in all three domains. In spite of thedifferences in gene expression cataloged here, however, the ideaof the gene itself is a unifying concept among all forms of life.

"/ R, A$P9,~m.:.\.\ ~ ii' '"r. ', DN..~1 ~/"". ~" ....

Poly~bosome

(~"PHA SIVENTEEN From Gene to Protein 347

DNA

3'

o Each amino aCidattaches to its proper tRNAwith the help of a specificenzyme and AlP.

TRANSLATION

••e <III

• ••

•

Q A succession of tRNA>add their amino acids tothe polypeptide chainas the mRNA is movedthrough the ribosomeone codon at a time.(When completed, thepolypeptide IS releasedfrom the ribosome.)

AminoacyHRNAsynthetase

Adivatedamino acid

tRNA

Aminoacid

Ribosome

Intron

RNA transcript(pre-mRNA)

\

~3'

............ RNApolymerase

NUCLEUS

o The mRNA leavesthe nucleus and

~",,",!.....:attta(heS 10 a ribosome.

RNA PROCESSING

e In eukaryotes, theRNA transcript (prernRNA) is spliced andmodified to producemRNA, which movesfrom the nucleus to thecytoplasm.

TRANSCRIPTION

5'

5'

CYTOPLASM

.... Figure 17.25 A summary oftranscription and translation in aeukaryotic cell. This diagram shows the pathfrom one gene to one polypeptide, Keep in mindthat each gene in the DNA (an be transcribedrepeatedly into many identical RNA molecules

arid that each mRNA can be translatedrepeatedly to yield many identical polypeptidemolecules. (Also, remember that the finalproduds of some genes are not polypeptides butRNA molecules, including tRNA and rRNA.) Ingerleral, the steps of trarlscription and translatiorl

are similar in bacterial, archaeal, and eukaryoticcells. The major difference is the occurrence ofRNA processing in the eukaryotic nucleus. Othersignificant differences are found in the initlatiorlstages of both transcription and trarlslation andin the termination of trarlscription.

348 UNIT THREE Gerlctks

Cater• .,JlRevlew

mRNA

-314 jf.M Go to the Study Area at www.masteringbio.com for BioFlix]-D Animations, MP3 Tutors, Videos, Practice Tests, an eBook, and more,

SUMMARY OF KEY CONCEPTS

."'1"1'-17.1Genes specify proteins via transcription andtranslation (pp. 325-331)

.. Evidence from the Study of Metabolic Defects DNAcontrols metabolism by directing cells to make specific en·zymes and other proteins. Beadle and Tatum's experimentswith lllutant strains of Neurospora supported the onegene-one enzyme hypothesis. Genes code for polypeptidechains or for RNA molecules.

.. Basic Principles ofTranscription and TranslationTranscription is the nucleotide-la-nucleotide transfer of information from DNA to RNA, while translation is the informational transfer from nucleotide sequence in RNA to aminoacid sequence in a polypeptide.

.. The Genetic Code Genetic information is encoded as a sequence of nonoverlapping base triplets, or codons. A codon inmessenger RNA (mRNA) either is translated into an aminoacid (61 of the 64 codons) or serves as a stop signal (3 codons).Codons must be read in the correct reading frame.

_M4if.MIn,'estigation How Is a Metabolic Pathway Analyz.ed?

MP3 Tutor DNA to RNA to ProteinActl\'lty Overview of Protein Synthesis

-"'1.01"-17.2Transcription is the DNA-directed synthesis of RNA:a closer look (pp. 331-334).. Molecular Components ofTranscription RNA synthesis

is catalyzed by RNA polymerase. It follows the same basepairing rules as DNA replication, except that in RNA, uracilsubstitutes for thymine.

Tran'iCnptlO/l unit

promoterl----~;~~~l--l

j:~~~~~rS' Template strand

RNA polymerase of DNA

_M4if.MActhity Transcription

-.11'''''-17.3Eukaryotic cells modify RNA after transcription(pp.334-336).. Alteration of mRNA Ends

Eukaryotic mRNA molecules areprocessed before leaving the nucleusby modification of their ends and byRNA splicing. The 5' end receives amodified nucleotide cap, and the 3'end a poly-A tail.

.. Split Genes and RNA Splicing Most eukaryotic genes haveintrons interspersed among the coding regions, the exons. InRNA splicing, introns are removed and exons joined. RNAsplicing is typically carried out by spliceosomes, but in somecases, RNA alone catalyzes its own splicing. The catalytic ability of some RNA molecules, called ribozymes, derives fromthe inherent properties of RNA. The presence of introns allows for alternative RNA splicing.

_MH'·MActh'ily RNA Processing

-"11• 01"-17.4Translation is the RNA-directed synthesis of apolypeptide: a closer look (pp. 337-344).. Molecular Components ofTranslation A cell translates an

mRNA message into protein using transfer RNAs (tRNAs).After binding specific amino acids, tRNAs line up via theiranticodons at complementary codons on mRNA. Ribosomeshelp facilitate this coupling with binding sites for mRNA andtRNA.

.. Building a Polypeptide Ribosomes m~NARiOOsomecoordinate the three stages of transla-tion: initiation, elongation, and termi-nation. The formation of peptide POlypeptide

bonds between amino acids is catalyzed by rRNA. A numberof ribosomes can translate a single mRNA molecule simultaneously, forming a polyribosome.

.. Completing and Targeting the Functional Protein Aftertmnslation, modifications to proteins can affect their threedimensional shape. Free ribosomes in the cytosol initiate synthesis of all proteins, but proteins destined for the endomembranesystem or for secretion are tmnsported into the ER. Such proteins have a signal peptide to which a signal-recognition particle(SRP) binds, enabling the translating ribosome to bind to the ER.

BloFlix 3-D Animation Protein Synthesis

Acthily Translation

Biology Labs On_Line Translationt ...b

-"1114"-17.5Point mutations can affect protein structure andfunction (pp. 344-346)

... Types of Point Mutations A point mutation is a change inone DNA base pair, which may lead to production of a nonfunctional protein. Base-pair substitutions can cause missense

CHAPTER SEVENTEEN From Gene to Protein 349

8. "'i"'i,,1I1 Review the roles of RNA by filling in the follOWing

table:

7. Which component is not directly involved in translation?

a. mRNA d. ribosomes

b. DNA e. GTP

c. tRNA

6. Which of the following mutations would be most likely to have

a harmful effect on an organism?

a. a base-pair substitution

b. a deletion of three nucleotides near the middle of a gene

c. a Single nucleotide deletion in the middle of an intron

d. a Single nucleotide deletion near the end of the coding

sequence

e. a Single nucleotide insertion downstream of, and dose to.

the start of the coding sequence

or nonsense mutations. Base-pair insertions or deletions mayproduce frameshift mutations.

.. Mutagens Spontaneous mutations can occur during DNAreplication, recombination, or repair. Chemical and physicalmutagens cause DNA damage that can alter genes.

_ •.1111"-17.6While gene expression differs among the domainsof life, the concept of a gene is universal(pp. 346-348).. Comparing Gene Expression in Bacteria, Archaea, and

Eukarya Because bacterial cells lack a nuclear envelope,translation can begin while transcription is still in progress.Archaeal cells show similarities to both eukaryotic and bacterial cells in their processes of gene expression. In a eukaryoticcell. the nuclear envelope separates transcription from translation, and extensive RNA processing occurs in the nucleus.

.. What Is a Gene? Revisiting the Question A gene is a region of DNA whose final functional product is either apolypeptide or an RNA molecule.

TESTING YOUR KNOWLEDGE

a.5'-UUUGGGAAA-3'

b.5'-GAACCCCTT·3'

c.5'-AAAACCTTT·3'

Type of RNA

Messenger RNA (mRNA)

d. 5'·CTTCGGGAA-3'

e. 5'·AAACCCUUU-3'

Functions

SELF-QUIZ

t. In eukaryotic cells, transcription cannot begin untila. the two DNA strands have completely separated and

exposed the promoter.b. several transcription factors have bound to the promoter.

c. the 5' caps are removed from the mRNA.

d. the DNA introns are removed from the template.

e. DNA nucleases have isolated the transcription unit.

2. Which of the following is not true of a codon?

a. It consists of three nucleotides.

b. It may code for the same amino acid as another codon.

c. It never codes for more than one amino acid.

d. It extends from one end of a tRNA molecule.

e. It is the basic unit of the genetic code.

3. The anticodon of a particular tRNA molecule is

a. complementary to the corresponding mRNA codon.

b. complementary to the corresponding triplet in rRNA.

c. the part of tRNA that bonds to a specific amino acid.

d. changeable. depending on the amino acid that attaches to

the tRNA.

e. catalytic, making the tRNA a ribozyme.

4. Which of the following is not true of RNA processing?

a. Exons are cut out before mRNA leaves the nucleus.

b. Nucleotides may be added at both ends of the RNA.

c. Ribozymes may function in RNA splicing.

d. RNA splicing can be catalyzed by spliceosomes.

e. A primary transcript is often much longer than the final

RNA molecule that leaves the nucleus.

5. Using Figure 17.5, identify a 5' ---> 3' se<juence of nucleotides in

the DNA template strand for an mRNA coding for the

polypeptide sequence Phe-Pro-Lys.


Transfer RNA (tRNA)

Plays catalytic (ribozyme) roles andstrUdural roles in ribosomes

Primary transcript

Small nuclear RNA (snRNA)

For Self-Qui: answers, see Appendix A.

_t,j4o!'.M Visit the Study Area at www.masteringbio.comforaPractice Test.

EVOLUTION CONNECTION

9. The genetic code (see Figure 17.5) is rich with evolutionary im

plications. For instance, notice that the 20 amino acids are not

randomly scattered; most amino acids are coded for by a simi

lar set ofcodons. What evolutionary explanations can be given

for this pattern? (Hint: There is one explanation relating to his

torical ancestry, and some less obvious ones of a "form-fits

function" type.)

SCIENTIFIC INQUIRY

10. Knowing that the genetic code is almost universal. a scientist

uses molecular biological methods to insert the human l3-globin

gene (shown in Figure 17.10) into bacterial cells. hoping the

cells will express it and synthesize functionall3-globin protein.

Instead, the protein produced is nonfunctional and is found to

contain many fewer amino acids than does l3-globin made by a

eukaryotic cell. Explain why.

Blologlcallnqulry: A W·orkbook oflm·estlgath·e Cases Explore translationand u~ of sequen,e data in testing hypothe~s with the ,a~ "The DoctorsDilemma,"

Chapter 17

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