AP Biology

Chapter 17 From Gene to Protein

AP Biology

What do genes code for?

proteins cells bodies

How does DNA code for cells & bodies? how are cells and bodies made from the

instructions in DNA

DNA

AP Biology

The “Central Dogma” Flow of genetic information in a cell

How do we move information from DNA to proteins?

transcriptiontranslation

replication

proteinRNADNA trait

AP Biology

Inheritance of metabolic diseases suggested that genes coded for enzymes each disease (phenotype) is caused by

non-functional gene product lack of an enzyme Tay sachs PKU (phenylketonuria) albinism

Metabolism taught us about genes

A B C D E

disease disease disease disease

enzyme 1 enzyme 2 enzyme 3 enzyme 4

metabolic pathway

AP Biology

Beadle & Tatum 1941 | 1958

George Beadle Edward Tatum

one gene : one enzyme hypothesisGenes dictate phenotypes through

enyzmes that catalyse specific chemical reaction in a cell

AP Biology

Wild-typeNeurospora

Minimalmedium

Select one ofthe spores

Grow oncomplete medium

Test on minimalmedium to confirmpresence of mutation

Growth oncompletemedium

X rays or ultraviolet light

asexualspores

spores

Minimal media supplemented only with…

• Beadle and Tatum causes bread mold to mutate with X-rays▫ Creating mutants that could not survive on minimal

medium▫ Arg-requiring mutants will use a pathway precursor

Ornithine → citrulline → arginine

AP Biology

Experiment

Class IMutants

Class IIMutants

Class IIIMutantsWild type

Minimal medium

(MM)(control)

MM +Ornithine

MM +Citrulline

MM +Arginine(control)

Gene A

1. Mutated spores will not grow on minimal medium due to a mutation in the pathway to produce arginine

2. This is a test to determine which gene was mutated in the pathway in each class of mutants

2. Any growth on minimal medium indicates where in the pathway the gene is mutated.

2. Ex: If the medium is supplemented with ornithine & growth occurs in all later tubes, one can conclude the gene that produces the enzyme to make ornithine was mutated.

Gene B

Gene C

AP Biology

CONCLUSION From the growth patterns of the mutants, Beadle and Tatum deduced that each mutant was unable to carry out one step in the pathway for synthesizing arginine, presumably because it lacked the

necessary enzyme. Because each of their mutants was mutated in a single gene, they concluded that each mutated gene must normally dictate the production of one enzyme. Their results supported the

one gene–one enzyme hypothesis and also confirmed the arginine pathway. (Notice that a mutant can grow only if supplied with a compound made after the defective step.)

Class IMutants(mutationin gene A)

Class IIMutants(mutationin gene B)

Class IIIMutants(mutationin gene C)Wild type

Gene A

Gene B

Gene C

Precursor Precursor Precursor Precursor

Ornithine Ornithine Ornithine Ornithine

Citrulline Citrulline Citrulline Citrulline

Arginine Arginine Arginine Arginine

EnzymeA

EnzymeB

EnzymeC

A A A

B B B

C C C

Conclusion

Gene A

Gene B

Gene C

AP Biology

The Products of Gene Expression: A Developing Story

As researchers learned more about proteins The made minor revision to the one gene–

one enzyme hypothesis Genes code for polypeptide chains or for

RNA molecules, not just enzymes Insulin, keratin, hemoglobin – 2 polypeptide

chains (2 genes)

AP Biology

mRNA

From gene to protein

DNAtranscription

nucleus cytoplasm

aa

aa

aa

aa

aa

aa

aa

aa

aa

aa

aa

protein

translation

ribosome

trait

AP Biology

Basic Principles of Transcription and Translation

Transcription Is the synthesis of RNA under the direction of DNA Produces messenger RNA (mRNA)

nucleus

Translation Synthesis of a polypeptide from instructions on mRNA Arranges monomers of polypeptide chain in their

linear order using the 20 different amino acids Occurs on ribosomes

DNA mRNA Protein

AP Biology

In prokaryotes Transcription and translation occur

together mRNA used immediately

Figure 17.3a

Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translated

without additional processing.

(a)

TRANSLATION

TRANSCRIPTION DNA

mRNA

Ribosome

Polypeptide

Eukaryotic cell. The nucleus provides a separatecompartment for transcription. The original RNA

transcript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA.

(b)

TRANSCRIPTION

RNA PROCESSING

TRANSLATION

mRNA

DNA

Pre-mRNA

Polypeptide

Ribosome

Nuclearenvelope

• In eukaryotes▫ RNA transcripts are modified

before becoming true mRNA

Prokaryotes v. eukaryotes

AP Biology

RNA ribose sugar N-bases

uracil instead of thymine U : A C : G

single stranded lots of RNAs

mRNA, tRNA, rRNA, siRNA…

RNADNAtranscription

AP Biology

Transcription Making mRNA

transcribed DNA strand = template strand synthesis of complementary RNA strand enzyme

RNA polymerase 2

template strand

rewinding

mRNA RNA polymerase

unwinding

DNAC C

C

C

C

C

C

C

C CC

G

GG

G

G G

G G

G

G

GAA

AA A

A

A

A

A

A A

A

AT

T T

T

T

T

T

T

T T

T

T

U U

5

35

3

3

5build RNA 53

AP Biology

Synthesis of an RNA: Transcription

The stages of transcription are Initiation Elongation Termination

PromoterTranscription unit

RNA polymerase

Start point

53

35

35

53

53

35

53

35

5

5

Rewound

RNA

RNA

transcript

3

3

Completed RNA transcript

Unwound

DNA

RNA

transcript

Template strand of DNA

DNA

1 Initiation. After RNA polymerase binds to

the promoter, the DNA strands unwind, and

the polymerase initiates RNA synthesis at the

start point on the template strand.

2 Elongation. The polymerase moves downstream, unwinding the

DNA and elongating the RNA transcript 5 3 . In the wake of

transcription, the DNA strands re-form a double helix.

3 Termination. Eventually, the RNA

transcript is released, and the

polymerase detaches from the DNA.

AP Biology

Initiation: Which gene is translated? Promoter region

binding site before beginning of gene TATA box binding site binding site for RNA polymerase

& transcription factors

Enhancer region binding site far

upstream of gene turns transcription

on HIGH

AP Biology

Transcription Factors Initiation complex

transcription factors bind to promoter region suite of proteins which bind to DNA “Transcription Initiation

Factors” turn on or off transcription Eukaryotes only

trigger the binding of RNA polymerase to DNA

AP Biology

Elongation RNA Pol II matches RNA bases to DNA

template strand 10-20 DNA bases exposed at a time Adds to 3’ end of a growing strand mRNA is synthesized from 5’ – 3’

U

A G GGGGGT T A C A C T T T T TC C C CA A

U

UU

U

U

G

G

A

A

A C CRNA

polymerase

C

C

C

C

C

G

G

G

G

A

A

A

AA

5' 3'

AP Biology

Termination of Transcription

Prokaryotes Have an end region

known as a terminator Termination region

causes dissociation of the RNA polymerase

Release mRNA for immediate use

Eukaryotes RNA Pol hits “polyadenylation signal

sequence” About 10-35 bases downstream,

proteins cut the growing RNA transcript free

Enzymes in eukaryotic nucleus modify the pre-mRNA before released into the cytoplasm

Alternations occur at the ends and interior sequences of the pre-mRNA

AP Biology

A A AA

A3' poly-A tail

mRNA

5'5' cap

3'

G PPP

50-250 A’s

More post-transcriptional processing Need to protect mRNA on its trip from

nucleus to cytoplasm Because enzymes in cytoplasm attack mRNA

protect the ends of the molecule add 5 GTP cap add poly-A tail at 3’ end (50-250 nucleotides)

longer tail, mRNA lasts longer: produces more protein Assists in ribosome attachment

UTR = untranslated region, includes 5’ cap and poly A tail

AP Biology

Eukaryotic genes have junk! Eukaryotic genes are not continuous

exons = the real gene expressed / coding DNA

introns = the junk inbetween sequence Noncoding sequences

eukaryotic DNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequence

intronscome out!

AP Biology

mRNA splicing

eukaryotic DNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequence

primary mRNAtranscript

mature mRNAtranscript

pre-mRNA

spliced mRNA

Post-transcriptional processing eukaryotic mRNA needs work after transcription primary transcript = pre-mRNA mRNA splicing

edit out introns make mature mRNA transcript

~10,000 bases

~1,000 bases

AP Biology

RNA splicing enzymes

snRNPs small nuclear RNA proteins

Spliceosome several snRNPs recognize splice

site sequence Every intron has a

short sequence at it’s end that is recognized

RNA transcript (pre-mRNA)

Exon 1 Intron Exon 2

Other proteinsProtein

snRNA

snRNPs

Spliceosome

Spliceosomecomponents

Cut-outintron

mRNA

Exon 1 Exon 2

5

5

5

1

2

3

AP Biology

Ribozymes Ribozymes

Are catalytic RNA molecules that function as enzymes and can splice RNA

Some organisms then can use their own RNA to complete splicing

AP Biology

The Functional and Evolutionary Importance of Introns

The presence of introns Can control gene activity Allows for alternative RNA splicing

Some genes are known to five rise to 2 or more different polypeptides depending on which segments are treated as exons during RNA processing

Ex: Sex determination in fruit flies Ex: The human genome functions with a relatively

small gene set Human Genome < Human proteome

AP Biology

Proteins consisting of discrete structural and functional regions called domains

Ex: one protein’s domain may be an enzymatic active site while another is for cell membrane attachment

In many cases Different exons code for the different domains in a protein Exon shuffling can result in

new proteins which hypothetically may lead to a beneficial variation

GeneDNA

Exon 1 Intron Exon 2 Intron Exon 3

Transcription

RNA processing

Translation

Domain 3

Domain 1

Domain 2

Polypeptide

Exon Shuffling

AP Biology

Translation

fromnucleic acid language

toamino acid language

AP Biology

How does mRNA code for proteins?

TACGCACATTTACGTACGCGGDNA

AUGCGUGUAAAUGCAUGCGCCmRNA

Met Arg Val Asn Ala Cys Alaprotein

?

How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?

AP Biology

AUGCGUGUAAAUGCAUGCGCCmRNA

mRNA codes for proteins in triplets

TACGCACATTTACGTACGCGGDNA

AUGCGUGUAAAUGCAUGCGCCmRNA

Met Arg Val Asn Ala Cys Alaprotein

?

codon

AP Biology

The code Code for ALL life!

strongest support for a common origin for all life

Code is redundant several codons for

each amino acid 3rd base “wobble”

Start codon AUG methionine

Stop codons UGA, UAA, UAG

Why is thewobble good?

AP Biology

How are the codons matched to amino acids?

TACGCACATTTACGTACGCGGDNA

AUGCGUGUAAAUGCAUGCGCCmRNA

aminoacid

tRNA anti-codon

codon

5 3

3 5

3 5

UAC

MetGCA

ArgCAU

Val

AP Biology

Translation: the basic concept

TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

Polypeptide

Aminoacids

tRNA withamino acidattachedRibosome

tRNA

Anticodon

mRNA

Trp

Phe Gly

A G C

A A A

CC

G

U G G U U U G G C

Codons5 3

AP Biology

Transfer RNA structure “Clover leaf” structure

Single RNA strand, about 80 nucleotides long anticodon on one end with complementary base pairs to mRNA codon amino acid attached on 3 end

AP Biology

Loading tRNA with correct amino acid Aminoacyl tRNA

synthetase enzyme which bonds

amino acid to tRNA bond requires energy

ATP AMP bond is unstable so it can release amino acid

at ribosome easily

AP Biology

Ribosomes Facilitate coupling of

tRNA anticodon to mRNA codon

Structure ribosomal RNA (rRNA) & proteins Made in eurkaryotic nucleolus 2 subunits

large small E P A

Ribosomes catalyze formation of peptide bonds between amino acids

AP Biology

Ribosomes

Met

5'

3'

UUA C

A G

APE

A site (aminoacyl-tRNA site) holds tRNA carrying next amino acid to

be added to chain P site (peptidyl-tRNA site)

holds tRNA carrying growing polypeptide chain

E site (exit site) empty tRNA

leaves ribosome from exit site

AP Biology

Building a polypeptide Initiation

brings together mRNA, ribosome subunits, initiator tRNA with start codon

Elongation adding amino acids based on codon

sequence

Termination stop codon on mRNA 123

Leu

Leu Leu Leu

tRNA

Met MetMet Met

PE AmRNA5' 5' 5' 5'

3' 3' 3'3'

U UA AAACC

CAU UG G

GUU

A AAAC

CC

AU UG GGU

UA

AAAC

CC

AU UG GGU U

A AACCA U UG G

G AC

ValSer

AlaTrp

releasefactor

AA A

CCU UGG 3'

Initiator tRNA with

start codon

AP Biology

Elongation of the Polypeptide Chain Amino acids are added one by one to the

preceding amino acid

Figure 17.18

Amino endof polypeptide

mRNA

Ribosome ready fornext aminoacyl tRNA

E

P A

E

P A

E

P A

E

P A

GDPGTP

GTP

GDP

2

2

site site5

3

TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

Codon recognition. The anticodon of an incoming aminoacyl tRNA

base-pairs with the complementary mRNA codon in the A site. Hydrolysisof GTP increases the accuracy and

efficiency of this step.

1

Peptide bond formation. An rRNA molecule of the large

subunit catalyzes the formation of a peptide bond between the

new amino acid in the A site and the carboxyl end of the growing

polypeptide in the P site. This step attaches the polypeptide to the

tRNA in the A site.

2

Translocation. The ribosome translocates the tRNA in the A

site to the P site. The empty tRNA in the P site is moved to the E site,

where it is released. The mRNA moves along with its bound tRNAs,

bringing the next codon to be translated into the A site.

3

AP Biology

Termination of Translation The final stage of translation is termination

When the ribosome reaches a stop codon in the mRNA

Figure 17.19

Release factor

Freepolypeptide

Stop codon(UAG, UAA, or UGA)

5

3 3

5

35

When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA.

1 The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed

from the ribosome.

2 3 The two ribosomal subunits and the other components of

the assembly dissociate.

AP Biology

Completing and Targeting the Functional Protein

Polypeptide chains Often are not immediately functional Undergo modifications after the translation process

Protein Folding and Post-Translational modifications occur After translation proteins may be modified in ways that affect their three-

dimensional shape

AP Biology

Polyribosomes

A number of ribosomes can translate a single mRNA molecule simultaneously Forming a polyribosome

Figure 17.20a, b

Growingpolypeptides

Completedpolypeptide

Incomingribosomalsubunits

Start of mRNA

(5 end)

End of mRNA

(3 end)

Polyribosome

An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes.

(a)

Ribosomes

mRNA

This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

0.1 µm(b)

AP Biology

Targeting Polypeptides to Specific Locations

Two populations of ribosomes are evident in cells Free and bound

Free ribosomes in the cytosol Initiate the synthesis of all proteins

Bound ribosomes make proteins of the endomembrane system and also proteins secreted from the cell Ex: insulin

AP Biology

Proteins to be secreted from the cell

Are marked with a signal peptide Transported to ER SRP (signal recognition protein) on ER

binds to signal peptide

start of a secretory pathway

AP Biology

Ribosome

mRNASignalpeptide

Signal-recognition

particle(SRP) SRP

receptorprotein

Translocationcomplex

CYTOSOL

Signalpeptide

removed

ERmembrane

Protein

ERLUMEN

Polypeptidesynthesis begins

on a freeribosome inthe cytosol.

An SRP binds to the signal

peptide, halting synthesis

momentarily.

The SRP binds to areceptor protein in the ERmembrane. This receptor

is part of a protein complex(a translocation complex)that has a membrane pore

and a signal-cleaving enzyme.

The SRP leaves, andthe polypeptide resumes

growing, meanwhiletranslocating across themembrane. (The signalpeptide stays attached

to the membrane.)

The signal-cleaving enzyme

cuts off thesignal peptide.

The rest ofthe completed

polypeptide leaves the ribosome andfolds into its final

conformation.

Summary

AP Biology

RNA plays multiple roles in the cell

Can hydrogen-bond to other nucleic acid molecules

Can assume a specific three-dimensional shape

Has functional groups that allow it to act as a catalyst (ribozymes)

AP Biology AAAAAAAAGTP

20-30b

3'

promoter transcriptionstop

transcriptionstart

introns

The Transcriptional Unit

transcriptional unit (gene)TAC ACT

DNA

DNATATA5'RNA

polymerase

pre-mRNA

5' 3'

translationstart

translationstop

mature mRNA

5' 3'

UTR UTR

exonsenhancer

1000+b

AP Biology 2007-2008

Protein Synthesis in Prokaryotes

Bacterial chromosome

mRNA

Cell wall

Cellmembrane

Transcription

AP Biology

Prokaryote vs. Eukaryote genes Prokaryotes

DNA in cytoplasm circular

chromosome Lack nuclear

envelope no introns

Eukaryotes DNA in nucleus linear

chromosomes DNA wound on

histone proteins introns vs. exons

eukaryoticDNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequence

AP Biology

Transcription & translation are simultaneous in bacteria DNA is in

cytoplasm no mRNA

editing ribosomes

read mRNA as it is being transcribed

Translation in Prokaryotes

AP Biology

Translation: prokaryotes vs. eukaryotes Differences between prokaryotes &

eukaryotes time & physical separation between

processes takes eukaryote ~1 hour

from DNA to protein no RNA processing

AP Biology

Can you tell the story?

AP Biology

Can you tell the story?

DNA

pre-mRNA

ribosome

tRNA

aminoacids

polypeptide

mature mRNA

5' GTP cap

poly-A tail

large ribosomal subunit

small ribosomal subunit

aminoacyl tRNAsynthetase

E P A

5'

3'

RNA polymerase

exon intron

tRNA

AP Biology

Changes in the genetic material of the cell

Mutations

AP Biology

When do mutationsaffect the nextgeneration?

Mutations Point mutations

single base change1. base-pair

substitution silent mutation

no amino acid change redundancy in code

missense change amino acid

nonsense change to stop codon

AP Biology

Point mutation leads to Sickle cell anemiaWhat kind of mutation?

Missense!

hydrophilicamino acid

hydrophobic amino acid

AP Biology

Mutations 2. Frameshift

shift in the reading frame

changes everything “downstream”

insertions adding base(s)

deletions losing base(s)

Where would this mutation cause the most change:

beginning or end of gene?

AP Biology

Deletion leads to Cystic fibrosis

loss of oneamino acid

delta F508

AP Biology

Mutagens Spontaneous mutations

Can occur during DNA replication, recombination, or repair Mutagens

Are physical or chemical agents that can cause mutations Ex: x-rays and radiation such as UV rays cause mutations Mutagens that cause cancer are carcinogens

Most mutagens are carcinogenic

AP Biology

What is a gene? revisiting the question

A gene Is a region of DNA whose final product is either a

polypeptide or an RNA molecule Some DNA makes rRNA, tRNA and other RNAs