Chem 45 Biochemistry: Stoker chapter 22 Nucleic Acids

Chapter 22

Nucleic Acids

Chapter 22

Table of Contents

Copyright © Cengage Learning. All rights reserved 2

22.1 Types of Nucleic Acids

22.2 Nucleotide Building Blocks

22.3. Nucleotide Formation

22.4 Primary Nucleic Acid Structure

22.5 The DNA Double Helix

22.6 Replication of DNA Molecules

22.7 Overview of Protein Synthesis

22.8 Ribonucleic Acids

22.9 Transcription: RNA Synthesis

22.10 The Genetic Code

22.11 Anticodons and tRNA Molecules

22.12 Translation: Protein Synthesis

22.13 Mutations

22.14 Nucleic Acids and Viruses

22.15 Recombinant DNA and Genetic Engineering

22.16 The Polymerase Chain Reaction

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Types of Nucleic Acids

Section 22.1


• The Swiss physiologist Friedrich Miescher (1844-1895) discovered

nucleic acids in 1869 while studying the nuclei of white blood cells.

• The fact that they were initially found in cell nuclei and are acidic

accounts for the name nucleic acid.

• It is now known that nucleic acids are found throughout a cell, not

just in the nucleus.

• Of all biomolecules, it is only the nucleic acids that have the

remarkable property of replicating itself, thus nature chose

these molecules to serve as the repository and transmitter of

genetic information in every cell and organism.

• The genome or total DNA of a cell acts like a molecular file where

the program for an organism’s activities (maintenance, development,

growth, reproduction, and even death) are encoded.


Section 22.1


• Cells in an organism are exact replicas

• Cells have information on how to make new cells

• Molecules responsible for such information are nucleic acids

• The nucleic acids (DNA in particular) are the “informational

molecules”; into their primary structure is encoded a set of directions

that ultimately governs the metabolic activities of the living cell.

• Two types of Nucleic Acids:

• DNA: Deoxyribonucleic Acid: found within cell nucleus

– storage and transfer of genetic information

– passed from one cell to other during cell division

• RNA: Ribonucleic Acid: occurs in all parts of cell

– primary function is to synthesize the proteins


Section 22.1


• Gene is a segment of DNA which specifies the chain of amino acids that comprises

the protein molecule

– most human genes are ~1000–3500 nucleotide units long

– genome: all of the genetic material (the total DNA) contained in the

chromosomes of an organism

– human genome is about 20,000–25,000 genes

• The genetic message is transcribed by mRNA and translated by tRNA and rRNA

into thousands of different proteins.

The Central Dogma

Section 22.2

Nucleotide Building Blocks


• Nucleic Acids: polymers in which

repeating unit is nucleotide

• A nucleotide has three components:

– pentose sugar - a

monosaccharide

– phosphate group (PO43-)

– heterocyclic base

Section 22.2



Nitrogen-Containing Heterocyclic Bases

Section 22.2



Section 22.3

Nucleotide Formation

Nucleoside Formation • Nucleoside: formed from condensation reaction between a five-carbon monosaccharide

and a purine or pyrimidine base derivative.

– the N9 of a purine or N1 of a pyrimidine base is attached to C1’ position of sugar (beta-

conformation) in an N-C-glycosidic linkage

• Nomenclature:

– for pyrimidine bases – suffix -idine is used (cytidine, thymidine, uridine)

– for purine bases – suffix -osine is used (adenosine, guanosine)

– prefix “-deoxy” is used to indicate deoxyribose present (e.g: deoxythymidine)


Section 22.3



• Phosphate group is

added to a nucleoside

– attached to C5’

position through a

phosphoester

bond

– condensation

reaction (H2O

released)

– named by

appending 5’-monophosphate to

nucleoside name


Section 22.3


Nucleotide Nomenclature


1) The 5’- nucleoside monophosphate of…is called….

a) adenosine…adenylic acid or adenosine

monophosphate (AMP)

b) guanosine…guanylic acid or guanosine

monophosphate (GMP)

c) cytidine…cytidylic acid or cytidine

monophosphate (CMP)

d) uridine…uridylic acid or uridine

monophosphate (UMP)

e) deoxythymidine…deoxythymidylic acid or

deoxythymidine monophosphate

(dTMP)

2) The 5’-nucleoside diphosphates are ADP,GDP,

CDP, UDP, dTDP

3) The 5’-nucleoside triphosphates are ATP, GTP,

CTP, UTP, dTTP

* If deoxyribose is present, the prefix deoxy is used

(dAMP, dADP, dATP, dGMP, dGDP, dGTP, dCMP,

dCDP, dCTP)

Section 22.3


Nucleotide Nomenclature


Section 22.4

Primary Nucleic Acid Structure

Primary Structure

• The nucleotides of a

polynucleotide chain are linked

to one another in 3’,5’-

phosphodiester bonds

• Phosphoric acid forms a

phosphate ester to connect

the 3’-hydroxyl group of one

pentose to the 5’-carbon on

another pentose

• Sugar-phosphate groups are

referred to as nucleic acid

backbone ; found in all

nucleic acids

• Sugars are different in DNA

and RNA


Section 22.4


Polynucleotides and the

Nucleic acids

• A ribonucleic acid (RNA) is a

polynucleotide in which each

of the monomers contains

ribose, a phosphate group,

and one of the heterocyclic

bases adenine, cytosine,

guanine, or uracil

• A deoxyribonucleic acid

(DNA) is a nucleotide polymer

in which each of the

monomers contains

deoxyribose, a phosphate

group, and one of the

heterocyclic bases adenine,

cytosine, guanine, or thymine.


Section 22.4


• 5’ end has free phosphate group and 3’

end has a free OH group

• the sequence of bases is read from 5’ to 3’

• the next nucleotide binds at the 3’ end


Section 22.4


Shorthand Structure of Polynucleotides • bases are indicated by their

initials, the ribose by a

straight line extending from

the base, and the phosphate

by P.

• the C3’ and C5’ of the ribose

or deoxyribose are indicated

by the fact that the C5’ is at

the end of the ribose line and

the C3’ is toward the middle

of the line.

• Takadiasase (mold)

attacks “b” linkages in which

“a” is linked to a purine

nucleotide

• RNAse (bovine pancreas)

attacks “b” linkages in which

“a” is linked to a pyrimidine

nucleotide


Section 22.5

The DNA Double Helix


• DNA, a high mol.wt., double-stranded

polynucleotide that occurs almost

exclusively in the nucleus of the cell

• primary function is storage and transfer of

genetic information which is used

(indirectly) to control many functions of a

living cell

• genetic information is encoded in the

primary structure of the DNA

• the primary structure of DNA is the

sequence of nucleotides in the chain

• the base content of DNA displays three sets

of equivalent pairs:

A + G = T + C (pu / pyr ratio = 1)

A = T

G = C

• the structure of the four bases permit

hydrogen bonding between specific base

pairs: Adenine always pairs with

Thymine and Guanine with Cytosine

Section 22.5



• the proof of this base-pairing came when

Watson and Crick proved by x-ray

diffraction that the DNA structure was a

double helix whose chains were

complementary and antiparallel

• complementary means that A binds

to T and C to G between the chains

- the sequence of bases on one

strand automatically determines the

sequence of bases on the other

strand

• antiparallel means that each end of

the helix contains the 5’ end of one strand

and the 3’ end of the other, so that the

chains travel in opposite directions

- only when the 2 strands are

antiparallel can the base pairs

form the H-bonds that hold them

together

Section 22.5



• The double helical secondary

structure of DNA is stabilized by a

number of factors:

• Chargaff’s rule of base pairing:

(A=T and GΞC)

%A = %T and %C = %G)

– example: human DNA

contains 30% adenine, 30%

thymine, 20% guanine and

20% cytosine

• Stacking interaction of the

hydrogen-bonded bases (the

purines and pyrimidine rings) at the

center

• Hydrophobic interior (bases) and

hydrophilic exterior (sugar-

phosphate backbone) ; contact with

bases through spiral grooves :

major and minor grooves

Section 22.5



Practice Exercise

• Predict the sequence of bases in the DNA strand

complementary to the single DNA strand shown below:

5’ A–A–T–G–C–A–G–C–T 3’

Section 22.5



Practice Exercise

• Predict the sequence of bases in the DNA strand

complementary to the single DNA strand shown below:

5’ A–A–T–G–C–A–G–C–T 3’

Answer:

3’ T–T–A–C–G–T–C–G–A 5’

Section 22.5



• the sugar-phosphate backbone of the two

strands spiral around the outside of the helix

like the handrails on a spiral staircase

• the nitrogenous bases extend into the center

at right angles to the acids of the helix as if

they are the steps of the spiral staircase

Denaturation of DNA

• The loss of helical structure due to disruption

of H–bonds is called denaturation or

melting, where the double strands separate

into single strands.

• This can be due to extremes of pH, heat, or

chemicals that disrupt H-bonds.

• DNAs which are G-C rich denature at a

higher temperature (Tm) than those which

are A-T rich.

Section 22.5



• Conformations of DNA:

• DNA can assume different conformations

because deoxyribose is flexible and the C–N-

glycosidic linkage rotates. (Recall that

furanose rings have puckered conformation)

• B-DNA – the common form as described by

Watson and Crick model

• A-DNA – when DNA becomes partially

dehydrated it assumes the A-form; observed

when DNA is extracted with solvents such as

ethanol.

• Z-DNA – named for its “zigzag” conformation;

DNA segments with alternating purine and

pyrimidine bases (esp. CGCGCG) are most

likely to adopt a Z configuration; regions of

DNA rich in GC repeats are often regulatory,

binding specific proteins that initiate or block

transcription.

Section 22.5



Types of DNA sequences:

1. Exons – the coding sequences; interrupted by

noncoding sequences

2. Introns – the noncoding sequences; from 10 to

10,000 bases long

3. Palindrome or inverted repeats

• a DNA sequence that contains the same

information whether it is read forward or

backward; e.g. “MADAM, I’M ADAM”

• tendency to form hairpin loop and a snapback

(cruciform)

• perfect palindrome forms with exact base pairs;

quasi palindrome, when not all will form hairpin

loop

4. Cruciform (or snapback)

• as their name implies, are crosslike structures

• when a DNA sequence contains a palindrome

Section 22.6

Replication of DNA Molecules


• Process by which DNA molecules

produce exact duplicates of themselves

• The two strands of the DNA double helix

unwind, the separated strands serve as

templates for the formation of new DNA

strands.

• Free nucleotides pair with the

complementary bases on the separated

strands of DNA.

• When the process is completed two

identical molecules of DNA are formed

• The newly synthesized DNA has one new

DNA strand and old DNA strand

• Two daughter DNA molecules are

produced from one parent DNA molecule,

with each daughter DNA molecule

containing one parent DNA strand and

one newly formed DNA strand.

Section 22.6



• DNA replication is semiconservative and mostly

bidirectional

• First step is the separation of the strands

– accomplished by helicase, which breaks

the H-bonds between base pairs

– positive supercoiling results when H-bonds

are broken, this is relieved by

topoisomerase

– when supercoiling is relieved, single-strand

binding protein binds to the separated

strands to keep them apart

– primase catalyzes synthesis of a 10-12

base piece of RNA to “prime” the DNA

replication

• DNA polymerase “reads” the parental strand

or template, catalyzing the polymerization of a

complementary daughter strand; the enzyme

checks the correct base pairing and catalyzes

the formation of phosphodiester linkages

Section 22.6



• There are different mechanisms for replication

of the two strands

• DNA polymerase enzyme can function only in

the 5’-to-3’ direction which can be offered only

by the 3’strand

1) the 3’ strand is called the leading strand

because it is replicated in a continuous

process in the direction of the

unwinding;

2) the 5’ strand is the lagging strand, it is

replicated in a discontinuous

mechanism and grows in segments

(Okazaki fragments) in the opposite

direction; the segments are later

connected by DNA ligase

In the Leading Strand:

• The DNA Polymerase, using dNTP’s and Mg2+,

cause the replication by base-pairing the

3’strand with free nucleotide units

Section 22.6



In the Lagging Strand

• The enzyme primase (using NTP’s and Mg2+)

puts primers on the lagging strand by forming

short RNA strands through base-pairing of the

5’strand.

• DNA Polymerase recognize, then lengthen

the primers using dNTP’s.

• The primers are then removed by

nucleotidase and further lengthening is done

by DNA Polymerase resulting to an OKAZAKI

STRAND.

• The Okazaki strands are then linked together

and sealed using the enzyme ligase leading to

the formation of a NEW STRAND

DNA replication usually occurs at multiple sites

within a molecule (origin of replication) and the

replication is bidirectional from these sites

• Multiple-site replication enables rapid DNA

synthesis

.

Section 22.6



Two conditions must be satisfied for replication to take place with high fidelity and accuracy:

a) normal electronic characteristics

b) normal base sequence

Section 22.7

Overview of Protein Synthesis


• Protein synthesis is directly under the

direction of DNA

• The expression of the information

contained in the DNA is fundamental to

the growth, development, and

maintenance of all organisms

• Proteins are responsible for the

formation of skin, hair, enzymes,

hormones, and so on

• Protein synthesis can be divided into

two phases.

– Transcription – a process by which

DNA directs the synthesis of mRNA

molecules

– Translation – a process in which

mRNA is deciphered to synthesize

a protein molecule

Section 22.8

Ribonucleic Acids


Differences Between RNA and DNA Molecules

• The sugar unit in the backbone of RNA is ribose; it is deoxyribose

in DNA.

• The base thymine found in DNA is replaced by uracil in RNA

• RNA is a single-stranded molecule; DNA is double-stranded

(double helix)

• A hairpin loop is produced when single-stranded RNA doubles

back on itself and complementary base pairing occurs.

• RNA molecules are much smaller than DNA molecules, ranging

from 75 nucleotides to a few thousand nucleotides

Section 22.8

Ribonucleic Acids


Types of RNA Molecules

• RNA functions primarily in the

synthesis of proteins, the molecules

that carry out essential cellular

functions

• Heterogeneous nuclear RNA (hnRNA)

- formed directly by DNA transcription.

• Messenger RNA (mRNA) - carries

instructions for protein synthesis

(genetic information) from DNA

• Small nuclear RNA (snRNA) -

facilitates the conversion of hnRNA to

mRNA.

• Ribosomal RNA (rRNA) - combines

with specific proteins to form

ribosomes - the physical site for

protein synthesis

• Transfer RNA (tRNA) - delivers amino

acids to the sites for protein synthesis

Section 22.9

Transcription: RNA Synthesis


Transcription

• biosynthesis of RNA by DNA-dependent RNA Polymerase on a DNA template; an information transfer

process where one of the two DNA strands acts as a template, which is copied into a complementary RNA

molecule

• transcription of DNA into RNA is restricted to discrete regions or genes of DNA

• when a gene is transcribed, only one strand of the DNA serves as the template for RNA synthesis: the

template strand, also called the sense strand; the nontemplate strand is called the coding strand, also

called the antisense strand.

• in the example below the blue 3’ strand is the template strand, or the sense strand. RNA polymerase

sythesizes an hnRNA (shown in green) in the 5' to 3' direction complementary to this template strand. The

opposite 5’ DNA strand (red), the nontemplate strand, is called the coding strand, or the antisense

strand. The easiest way to find the corresponding hnRNA sequence (shown in green) is to read the coding,

or antisense strand directly in the 5' to 3' direction substituting U for T.

5' T G A C C T T C G A A C G G G A T G G A A A G G 3'

3' A C T G G A A G C T T G C C C T A C C T T T C C 5'

5' U G A C C U U C G A A C G G G A U G G A A A G G 3'

Section 22.9



Steps in the Transcription Process

• RNA Polymerase recognizes promoter

sites (sequence of bases which signals

where to start) and enhancer sites (base

sequence which make recognition clearer)

on DNA.

• These sites interact to define the region

for transcription.

• Like DNA Polymerase, RNA Polymerase

requires a 5’-3’ direction which can only

be provided by the 3’strand of DNA

• The RNA Polymerase, once it has spotted

the portion to be transcribed, does the

transcription in the 5’ 3’ direction and

uses NTP’s and Mg2+

Section 22.9



Steps in the Transcription Process

• Unwinding of DNA double helix by RNA

polymerase to expose some bases (a

gene):

• Alignment of free ribonucleotides along

the exposed DNA strand (template)

forming new base pairs

• RNA polymerase catalyzes the linkage of

ribonucleotides one by one to form

hnRNA molecule

• Transcription ends when the RNA

polymerase enzyme encounters a stop

signal on the DNA template:

• The newly formed hnRNA molecule and

the RNA polymerase are released

• Transcription occur with very high

accuracy and fidelity (normal base

sequence and normal electronic

character) except under conditions of

spontaneous and induced mutation

Section 22.9



Post-Transcription Processing: Formation of mRNA • hnRNA is a primary transcript which is

processed in post-transcriptional

modification, a three step process:

– A 5' cap structure is added; this

structure is required for efficient

translation of the final mRNA

– A 3' poly(A) tail is added by poly(A)

polymerase to protect the 3' end of

the mRNA from enzymatic digestion;

prolongs the life of the mRNA

– RNA splicing removes portions of

the primary transcript that are not

protein coding

• transcripts due to introns are removed

by spliceosomes, composed of “small

nuclear ribonucleoproteins (snRNPs,

read “snurps”) ; the transcripts due to

exons are joined by ligase

• the exons in DNA are transcribed as the

codons in mRNA

Section 22.9



• Once the mRNA is formed and released from DNA, it moves into the cytoplasm

and combines with rRNA in ribosomes where protein synthesis occurs.

Section 22.10

The Genetic Code


• In translation, the base

sequence in mRNA

determines the amino

acid sequence of the

protein synthesized

• The base sequence of

an mRNA molecule

involves only 4

different bases - A, C,

G, and U

• The code is a triplet

code since it involves

3 bases per coding

unit.

• The coding unit is

called a codon.

• The genetic code is a

series of base triplets

in mRNA called

codons that code for

a particular amino

acid.

Section 22.10

The Genetic Code


• The genetic code is highly

degenerate:

– many amino acids are

designated by more than one

codon.

– Arg, Leu, and Ser are

represented by six codons.

– most other amino acids are

represented by two codons

– Met and Trp have only a

single codon.

– codons that specify the same

amino acid are called

synonyms

• There is a pattern to the

arrangement of synonyms in the

genetic code table.

– all synonyms for an amino

acid fall within a single box

unless there are more than

four synonyms

Section 22.10

The Genetic Code


• The genetic code is almost

universal:

– with minor exceptions the

code is the same in all

organisms

– the same codon specifies

the same amino acid

whether the cell is a

bacterial cell, a plant cell,

or a human cell.

• An initiation codon exists:

– the existence of “stop”

or termination codons

(UAG, UAA, and UGA)

suggests the existence

of “start” codons.

– the codon - coding for

the amino acid

methionine (AUG)

functions as initiation

codon.

Section 22.10

The Genetic Code

Practice Exercise

Sections A, C, and E of the following base sequence section of a

DNA template strand are exons, and sections B and D are

introns.

a. What is the structure of the hnRNA transcribed from this

template?

b. What is the structure of the mRNA obtained by splicing the

hnRNA?


Section 22.10

The Genetic Code

Practice Exercise

Sections A, C, and E of the following base sequence section of a

DNA template strand are exons, and sections B and D are

introns.

a. What is the structure of the hnRNA transcribed from this

template?

b. What is the structure of the mRNA obtained by splicing the

hnRNA?


Answers:

a. 3’ GCG–GCA–UCA–ACC–GGG–CCU–CCU 5’

b. 3’ GCG–ACC–CCU–CCU 5’ or 5’ UCC-UCC-CCA-GCG

3’

Section 22.10

The Genetic Code

Practice Exercise

The structure of an mRNA segment obtained from a DNA

template strand is


mRNA 3’ ACG-AGC-CCU-CUU 5’

What polypeptide amino acid sequence will be synthesized using

this mRNA?

Section 22.10

The Genetic Code

Practice Exercise

The structure of an mRNA segment obtained from a DNA

template strand is


mRNA 3’ ACG-AGC-CCU-CUU 5’

What polypeptide amino acid sequence will be synthesized using

this mRNA?

Answer: Phe-Ser-Arg-Ala

Section 22.11

Anticodons and tRNA Molecules


• During protein synthesis amino

acids do not directly interact

with the codons of an mRNA

molecule.

• tRNA molecules as

intermediates deliver the amino

acids to mRNA.

• Two important features of tRNA

– the 3’end is where an

amino acid is covalently

bonded to the tRNA.

– the loop opposite to the

open end is the site for a

sequence of three bases

called an anticodon.

• Anticodon - a three-nucleotide

sequence on a tRNA molecule

that is complementary to a

codon on an mRNA molecule.

• Codon-anticodon interaction is

antiparallel

Section 22.12

Translation: Protein Synthesis

Practice Exercise


Section 22.12


Practice Exercise

A tRNA molecule possesses the anticodon 5’ CGU 3’ . Which

amino acid will this tRNA molecule carry?


Section 22.12


Practice Exercise

A tRNA molecule possesses the anticodon 5’ CGU 3’ . Which

amino acid will this tRNA molecule carry?


Answer: Thr

Section 22.12



• Translation – a process in which mRNA

codons are deciphered to synthesize a

protein molecule

• Ribosome – an rRNA–protein complex

that serves as the site, or “workbench” for

protein synthesis:

• Ribosomal RNA (rRNA)

– constitutes about 60% of the

ribosomes (40% protein)

– structurally composed of two spherical

particles of unequal size: the smaller

has affinity for mRNA ; the larger has

an attraction for tRNA ;

– has two sites to bind tRNA

• P-site binds to the growing

peptide

• A-site binds the aminoacyl tRNA

Section 22.12



Five Steps of Translation Process

• Activation of tRNA: addition of specific amino acids to the 3’-OH

group of tRNA.

• Initiation of protein synthesis: Begins with binding of mRNA to small

ribosomal subunit such that its first codon (initiating codon AUG)

occupies a site called the P site (peptidyl site)

• Elongation: Adjacent to the P site in an mRNA–ribosome complex

is A site (aminoacyl site) and the next tRNA with the appropriate

anticodon binds to it. Peptidyl transferase links the A site and P site

amino acids via a peptide bond.

• Termination: The polypeptide continues to grow via translocation

until all necessary amino acids are in place and bonded to each

other. The process stops when a stop codon is encountered.

• Post-translational processing: Gives the protein the final form it

needs to be fully functional

Section 22.12




• (1) Activation of tRNA: addition of

specific amino acids to the 3’-OH group

of tRNA.

• The amino acid combines with a

molecule of ATP, yielding a compound

known as aminoacyl adenylate.

• The reaction is enzyme-catalyzed

• The aminoacyl adenylate remains on the

surface of the enzyme and then

undergoes reaction with the proper

tRNA molecule to form the

corresponding aminoacyl-tRNA

complex (charged tRNA or activated

tRNA).

Section 22.12




• (2) Initiation of protein

synthesis begins with the

formation of an initiation

complex

• mRNA binds to small

ribosomal subunit such

that its first codon

(initiating codon AUG)

occupies a site called the

P site (peptidyl site)

• The initiator tRNA

recognizes the initiation

codon, AUG.

• The large ribosomal

subunit binds to form the

complete, functional

ribosome.

Section 22.12




• (3) Elongation occurs in three steps that are repeated until protein

synthesis is complete:

– (3a), the binding of the aminoacyl-tRNA to the empty A-site (amino acyl-

tRNA binding site)

– (3b), peptide bond formation occurs catalyzed by an enzyme peptidyl

transferase that is part of the ribosome. Now the peptide chain is shifted

to the tRNA that occupies the A site.

– (3c), the uncharged tRNA molecule left on the P site is discharged, and

the ribosome changes position so that the next codon on the mRNA

occupies the A-site. This movement is called translocation, which shifts

the new peptidyl-tRNA from the A-site to the P-site.

Section 22.12




• (3) Elongation occurs in three

steps that are repeated until protein

synthesis is complete:

– (3a), the binding of the

aminoacyl-tRNA to the empty

A-site (amino acyl-tRNA binding

site)

– (3b), peptide bond formation

occurs catalyzed by an enzyme

peptidyl transferase that is part

of the ribosome.

– Now the peptide chain is shifted

to the tRNA that occupies the A

site.

Section 22.12




• The transfer of

an amino acid

(or growing

peptide chain)

from the P site

to the A site

during peptide

bond formation

is an example

of an acyl

transfer

reaction

Section 22.12




• (3) Elongation occurs in three

steps that are repeated until

protein synthesis is complete:

– (3c), the uncharged tRNA

molecule left on the P site is

discharged, and the ribosome

changes position so that the

next codon on the mRNA

occupies the A-site.

– This movement is called

translocation, which shifts the

new peptidyl-tRNA from the

A-site to the P-site.

Section 22.12



Five Steps of Translation

• (4) Termination: The

polypeptide continues to grow

via translocation until all

necessary amino acids are in

place and bonded to each other.

The process stops when a stop

codon is encountered.

• (5) Post-translational

processing: Gives the protein

the final form it needs to be fully

functional

– cleavage of f-met (initiation

codon); association with

other proteins; bonding to

carbohydrate or lipid groups;

S – S bonds between cys

units

Section 22.12



Section 22.12



Efficiency of mRNA Utilization

• Polysome (polyribosome):

The complex of a mRNA

and several ribosomes

• Many ribosomes can move

simultaneously along a

single mRNA molecule

• The multiple use of mRNA

molecules reduces the

amount of resources and

energy that the cell

expends to synthesize

needed protein

• In the process – several

ribosomes bind to a single

mRNA - polysomes

Section 22.12



• Since protein is not synthesized continuously but only as needed, the DNA

must normally be in a “repressed” state

• A repressor, which is a polypeptide, binds to small segment of the DNA

called the operator site

• As long as the repressor is bonded to the operator site of the DNA, no

mRNA is produced and protein synthesis is inhibited

• When a particular protein is needed, an inducer is formed

• The inducer combines with the repressor changing its shape so that it can

no longer bind to the DNA

• Once the repressor is removed from the DNA, synthesis of mRNA and

hence, protein can begin

• When sufficient protein has been synthesized, the inducer is removed and

the repressor once again binds the DNA, stopping protein synthesis

Regulation of protein synthesis

Section 22.12



Antibiotics inhibit bacterial protein synthesis

Antibiotic Effect on ribosomes to inhibit protein synthesis

Chloramphenicol Inhibits peptide bond formation and prevents the binding of tRNA’s

Erythromycin Inhibits peptide chain growth by preventing the translocation of the

ribosome along the mRNA

Puromycin Causes release of an incomplete protein by ending the growth of the

polypeptide early

Streptomycin Prevents the proper attachment of tRNA’s; mRNA misreading by binding

30S

Tetracycline Prevents the binding of tRNA’s by binding to 30S subunit

========================================================================

Several antibiotics stop bacterial infections by interfering with the synthesis

of proteins needed by the bacteria. Some antibiotics act only on bacterial

cells by binding to the ribosomes in bacteria, but do not act on human cells.

Section 22.13

Mutations


Replication, transcription, and translation occur with very high accuracy

and fidelity (normal base sequence and normal electronic character)

except under conditions of spontaneous and induced mutation

• An error in base sequence reproduced during DNA

replication

• Errors in genetic information is passed on during

transcription.

• The altered information can cause changes in amino

acid sequence during protein synthesis and thereby alter

protein function

• Such changes have a profound effect on an organism.

Section 22.13

Mutations


Spontaneous Mutations

1. Point Mutations

- substitution of a single nucleotide for

another caused by tautomeric base-

mispairs due to the ease by which rare

tautomers are formed

- C is the most mutable base due to the

very small energy difference between its

two tautomers

- in nature, there are more A-T pairs than

G-C pairs to protect us from the effect of

spontaneous mutation

A. Transition (C-A* ; G-T* ; C *- A mispair)

- a purine base is changed to another

purine; a pyrimidine base to another

pyrimidine

B. Transversion (A-A* mispair)

- a purine base is changed to pyrimidine;

a pyrimidine base changed to purine

Section 22.13

Mutations


Spontaneous Mutations

2. Frameshift mutation

-leads to a change in the

reading frame

A. Insertion

B. Deletion

In an insertion or deletion

mutation, one or more

nucleotides are added to or

deleted from the DNA

sequence.

Then a frameshift occurs

which leads to a misreading

of all the codons following

the base change.

Section 22.13

Mutations


Section 22.13

Mutations


Mutagens

• Although a number of structural features of nucleic acids promote

stabilization of base sequences, reactivity with some physical and chemical

agents can alter the electronic characteristics of the bases and other

structural units.

• Consequently, nucleic acid functions would be affected

• A mutagen is a substance or agent that causes a change in the structure of

a gene:

– Physical agents : heat, Ultraviolet, ionizing radiation (X-ray, gamma

rays)

– Chemical agents :HNO2 can convert cytosine to uracil

• Nitrites, nitrates, and nitrosamines – can form nitrous acid in cells

• Under normal conditions mutations are repaired by repair enzymes

Section 22.13

Mutations


Induced Mutations – Physical Agents

• UV radiation

• absorbed primarily by the pyrimidine bases.

– UV light causes covalent linkage of

adjacent pyrimidine bases forming

cyclobutane pyrimidine dimer

– when T is irradiated with UV, excitation of

pi electrons to antibonding MO’s will result

in the formation of T diradicals. Coupling

of T diradicals may result in the formation

of thymine cyclobutane dimers

– Failure to repair this defect can lead to

xeroderma pigmentosum; people who

suffer from this genetic skin disorder are

very sensitive to UV light and develop

multiple skin cancers

– No purine dimers since purines are more

thermodynamically stable than

pyrimidines

Section 22.13

Mutations


Induced Mutations – Physical Agents

• heat mutagenesis

- characterized by transmigration of N – C

glycosidic bonds producing neoguanosine

crosslinks

• ionizing radiation

– more often when a plant or animal is

irradiated most of the energy is deposited

in the aqueous phase. Less often will a

primary ionization occur in an organic

molecule

– a portion of damage to the living system

results from reactive particles that are

formed in the water phase and diffuse to

an organic molecule in the cell causing

secondary reactions (free radicals are

implicated in radiation damage)

Section 22.13

Mutations


Induced Mutations – Chemical Agents

• intercalating agents (PAH), alkylating agents, heavy metal ions, etc.

• one notorious source of numerous mutagens is cigarette smoke

• It contains PAH, nitrosamines, hydrazines, pyrolysates, alkoxy free

radicals, superoxide anion radicals, Cd2+, etc.

• other notorious sources are: cured foods; burnt portion of broiled

fish and meat; moldy peanuts and cereals; pesticides;

• polluted air (epoxides, SO2, ozone, Pb2+, ethylene dibromide, etc.)

• laboratory chemicals (benzene has been linked to leukemia, CHCl3,

CCl4, etc)

Section 22.13

Mutations



• Intercalating agents

- with polycyclic planar structures, like PAH,

e.g. benzo(a)pyrene, interpose between the

strands within the grove of DNA

- inhibit its replication/transcription, or cause

deletions

Section 22.13

Mutations



• Alkylating agents

- have electrophilic sites or may be

metabolized to electrophiles which

can interact with alkylating sites at

DNA; include PAH, nitrosamines,

aflatoxins, aromatic amines,

epoxides, nitrogen mustard,

nitrosoureas, etc.

- bases in DNA are nucleophiles and

as such are strongly attracted to

electrophilic compounds

• N7- alkylation leads to apurinic sites

(positivity of R is relayed to C8 & N9 thereby

enhancing the dipositivity of the N-C –

glycosidic bond & render it more less

stable)

• O6- alkylation leads to base mispairs

Section 22.13

Mutations


Chemical mutagens. (a) HNO2

(nitrous acid) converts cytosine

to uracil and adenine to

hypoxanthine. (b) Nitrosoamines,

organic compounds that react to

form nitrous acid, also lead to

the oxidative deamination of A

and C. (c) Hydroxylamine

(NH2

OH) reacts with cytosine,

converting it to a derivative that

base-pairs with adenine instead

of guanine. The result is a C-G

to T-A transition. (d) Alkylation of

G residues to give O6

-

methylguanine, which base-pairs

with T. (e) Alkylating agents

include nitrosoamines,

nitrosoguanidines, nitrosoureas,

alkyl sulfates, and nitrogen

mustards. Note that

nitrosoamines are mutagenic in

two ways: they can react to yield

HNO2

or they can act as

alkylating agents.

Section 22.13

Mutations


When DNA is hit by a mutagen

Lesions repaired Cell death

Mutagen

Cells during Organogenesis

in in in

Somatic cells

Lesions escape repairs

Germ cells

CANCER BIRTH DEFECTS

STERILITY

GENETIC DISORDER

(can be transmitted from

one generation

to the next)

DNA

Section 22.13

Mutations


Some chemical and environmental carcinogens

Carcinogen Tumor occurrence

Asbestos Lung, respiratory tract

Arsenic Skin, lung

Cadmium Prostate, kidneys

Chromium Lung

Nickel Lung, sinuses

Aflatoxin Liver

Nitrites Stomach

Aniline dyes Bladder

Vinyl chloride Liver

Benzene Leukemia

Section 22.13

Mutations


DNA Repair Mechanisms

• DNA is the only macromolecule that is repaired

rather than degraded.

• The repair processes are very efficient with fewer

than 1 out of 1,000 accidental changes resulting

in mutations.

• The rest are corrected through various repair

mechanisms before, during, or after replication

A) photoreactivation repair

uses an enzyme photolyase, which

binds the T-T cyclobutane dimer & in

the presence of visible light changes

the cyclobutane ring back into

individual pyrimidine bases

Section 22.13

Mutations


DNA Repair Mechanisms

B) excision repair

mutations are excised by a

series of enzymes that remove

incorrect bases and replace

them with the correct ones.

Base excision repair – involves a

battery of enzymes called DNA

glycosylases each of which recognizes a

single type of altered base in DNA and

catalyzes its hydrolytic removal from

the deoxyribose sugar (e.g., removing

deaminated cytosine, deaminated

adenine, alkylated bases, etc.)

Nucleotide excision repair

Section 22.14

Nucleic Acids and Viruses


Viruses

• Viruses: Tiny disease causing agents with outer protein

envelope and inner nucleic acid core

• They can not reproduce outside their host cells (living

organisms)

• Invade their host cells to reproduce and in the process

disrupt the normal cell’s operation

• Virus invade bacteria, plants animals, and humans

– Many human diseases are of viral origin, e. g. Common cold,

smallpox, rabies, influenza, hepatitis, and AIDS

Section 22.14

Nucleic Acids and Viruses


Human cancers caused by oncogenic viruses

Virus Disease

RNA viruses

Human T-cell leukemia-lymphoma virus-1 Leukemia

Human immunodeficiency virus Acquired immune deficiency (AIDS)

DNA viruses

Epstein-Barr virus Burkitt’s lymphoma (cancer of wbc)

Nasopharyngeal carcinoma

Hodgkin’s disease

Hepatitis B virus Liver cancer

Herpes simplex virus Cervical and uterine cancer

Papilloma virus Cervical and colon cancer, genital warts

Section 22.15

Recombinant DNA and Genetic Engineering


• Recombinant DNA: DNA molecules that have been

synthesized by splicing a sequence of segment DNA

(usually a gene) from one organism to the DNA of

another organism.

• Genetic Engineering (Biotechnology): A process in

which an organism is intentionally changed at the

molecular (DNA) level so that it exhibits different traits.

Section 22.15



• First genetically engineered organism are bacteria

(1973) and Mice (1974)

• Insulin producing bacteria - commercialized in 1982.

– Bacteria act as protein factories

• Many plants have now been genetically engineered and

numerous beneficial situations have been created.

– Disease resistance – increased crop yield

– Drought resistance – consumption of less water

– Predator resistance – less insecticide use

– Frost resistance – resist changes in temps below freezing.

– Deterioration resistance – long shelf-life.

Benefits

Section 22.15



Recombinant DNA Production using a Bacterial Plasmid

• Dissolution of cells:

– E. coli cells of a specific strain containing the plasmid of interest are treated with

chemicals to dissolve their membranes and release the cellular contents

• Isolation of plasmid fraction:

– The cellular contents are fractionated to obtain plasmids

• Cleavage of plasmid DNA:

– Restriction enzymes are used to cleave the double-stranded DNA

• Gene removal from another organism:

– Using the same restriction enzyme the gene of interest is removed from a

chromosome of another organism

• Gene–plasmid splicing:

– The gene (from Step 4) and the opened plasmid (from Step 3) are mixed in the

presence of the enzyme DNA ligase to splice them together.

• Uptake of recombinant DNA:

– The recombinant DNA prepared in step 5 are transferred to a live E. coli culture

where they can be replicated, transcribed and translated.

Section 22.15



• Transformed cell can reproduce a large number of

identical cells –clones:

– Clones are the cells that have descended from a

single cell and have identical DNA

• Given bacteria grow very fast, within few hours 1000s of

clones will be produced

• Each clone can synthesize the protein directed by

foreign gene it carries

Section 22.15



Recombinant DNA Production using a Bacterial Plasmid

Section 22.16

The Polymerase Chain Reaction


• The polymerase chain reaction (PCR): A method for rapidly

producing multiple copies of a DNA nucleotide sequence (gene).

• This method allows to produce billions of copies of a specific gene

in a few hours.

• PCR is very easy to carryout and the requirements are:

– Source of gene to be copied

– Thermostable DNA polymerase

– Deoxynucleotide triphosphates (dATP, dGTP, dCTP and dTTP)

– A set of two oligonucleotides with complementary sequence to

the gene (primers)

– Thermostable plastic container and

– Source of heat

Section 22.16

The Polymerase Chain Reaction


Chem 45 Biochemistry: Stoker chapter 22 Nucleic Acids

Education

Transcript of Chem 45 Biochemistry: Stoker chapter 22 Nucleic Acids