Genetics Chapter 11

8/13/2019 Genetics Chapter 11

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Gene Expression: Translation

Chapter 11



DNA directs the synthesis of proteins

• concept that genes control enzymes

• structure of proteins

• the mechanism of translation

- prokaryotes, eukaryotes

• post-translational modifications to polypeptides

• the genetic code



Genes control enzymes : Garrod

1. Archibald Garrod (1909):

- individuals with particular homozygous alleles expressed

particular disease, including alkaptonuria and albinism

a defect in a single gene can result

in a metabolic deficiency that causes

an identifiable phenotypic condition



Genes control enzymes : Beadle and Tatum

Concluded that one gene encoded one enzyme (1941)

current hypothesis: each gene encodes 1 enzyme (or protein)

that functions in a cell to determine a phenotype

Isolated mutant strains of Neurospora that could only grow if

niacin was supplied in culture medium (auxotrophs)- hypothesis : each strain is deficient in 1 enzyme in niacin

pathway

Different intermediates were fed to mutant strains of Neurospora

this positioned the mutants in the niacin biosynthetic pathway



One enzyme can impact more than

one phenotype

Pleiotropy• Defect in a single gene affects one step in a

biochemical pathway but has multiple effects

- example :mutation that causes tyrosinosis

prevents the degradation of phenylalanine

and tyrosine however, may result in multiple symptoms

(phenotypes): ulcers in corneas, lesion on

skin, mental retardation



Proteins Are Composed of Amino

Acids

• Amino (NH2) group

• Carboxyl (COOH) group

• Side chains (R groups), four classes:

1. Acidic

2. Basic

3. Nonpolar (hydrophobic)

4. Polar (uncharged)



The 20 common amino acids

* all side chains are shown at physiological pH (6.8)



Joining amino acids

Amino acids are joined together by peptide bonds

“dehydration synthesis”

dipolar ion

(zwitterion)



Protein Structure• Primary: Linear sequence of amino acids

• Secondary: Common folding patterns that form dueto maximization of hydrogen bonds (alpha helix,beta pleated sheet) in peptide backbone of nearby

amino acids (others : extended strands, turns,random coils)

• Tertiary: Overall three-dimensional structure of

protein

• Quaternary: Interaction of more than onepolypeptide to form active protein

** each level of structure depends on the level below it



Secondary Structure

-helix

-pleated sheet



Tertiary Structure

Three-dimensional structure ofhuman -globin

forms active site

of an enzyme



Quaternary Structure

human hemoglobin

-globin

-globin



Translation Occurs on the Ribosome• Codons (3 base sequences) in mRNA are read sequentially

• tRNAs read the codons using their anticodons• tRNAs have amino acids attached to their 3’ ends

• Amino acids are joined by peptide bonds

• sequence of amino acids in protein is specified by sequence of

codons in mRNA

E. co li

ribosome

A : aminoacyl site

P : peptidyl siteE : exit site



Aminoacyl-tRNA synthetases

• Covalently attaches (charges) the correct amino acid to 3’ end of the

correct tRNA : recognizes acceptor s tem and ant icodon of tRNA

• Charging is a two-stage reaction in active site of tRNA synthetase:

1. amino acid reacts with ATP (binds AMP, releases PPi)

2. amino acid is detached from AMP and joined to 3’ end of the tRNA



Decoding Machinery

• 20 different aminoacyl tRNA synthetases

• More than 20 tRNAs

• 64 codons (61 code for a.a. , 3 for STOP codons)

• anticodon of tRNA, and not the amino acid itself,

determines which amino acid is incorporated

result : Ala incorporated into protein

anticodon on tRNA, not amino

acid, is recognized and

dictates which amino acid

is incorporated

anticodon anticodon



Translation

• Initiation

- formation of ribosome

- recognition of 1st codon

- positioning 1st charged tRNA at P site

• Elongation

- add a.a. from charged tRNAs at A site

• Termination

- recognize STOP codon- stop elongation

- release of polypeptide



Translational Initiation Must Start

at Correct Codon

• Frameshift mutations: deletion or insertion of one or morebases in mRNA, resulting in shift of reading frame

- If ribosome shifts out of frame it translates different codons,

resulting in a nonfunctional protein



Initiator methionine in E. coli

(initiator methionine)(internal methionine)



Initiation of Translation in E. coli

(N-formyl-methionine)

binds at

P site of 30S

IF2 only binds fMet

fMet is the only initiator aa

E : exit site

P : peptidyl tRNA site

A : aminoacyl tRNA site

IF1 binds at A site, prevents 1st

tRNA from binding there

I iti ti f T l ti i E li



Initiation of Translation in E.coli :

Shine-Dalgarno sequence in mRNA

base pairs with16S rRNA

16S rRNA

mRNA (just upstream of initiator AUG)





• eIF3 binds 40S subunit

• eIF2-GTP binds Met-tRNA

and then binds 40S subunit

• eIF4 binds 5’ Cap of mRNA

• 5’ Cap of mRNA binds

complex of 40S subunit,

eIF2-GTP, eIF3, and

Met-tRNA

Initiation of Translation in Eukaryotes

Initiator complex forms at 5’ cap :

i

Met

i

Met

GTP

GTP

GTP

PABP i t ti ith i iti t l



PABP interactions with initiator complexes

enhance translation by helping initiation and

stabilizing mRNApolyA binding protein

http://www.nature.com/horizon/rna/highlights/figures/s2_spec1_f1.html

may also insure that only mature mRNAs are translated

f



Initiation of Translation in Eukaryotes:

Finding the Start Codon (AUG)

• binding of eIF1 and eIF1Astimulates complex to

scan for AUG

• eIF2-GTP hydrolyzes GTP,

then eIF2-GDP and eIF3

leave complex

• eIF5 allows large

subunit (60S) to join complex

GTP

GTP

GTP

-GDP



Role of eIF2 proteins

• eIF2 required to bring met-tRNA to small

ribosomal subunit

• eIF2B required to reconstitute the active form

of eIF2 (eIF2-GTP) from eIF2-GDP

i

Met

Mechanisms to Identify Start




Codon AUG

Scanning Model

ribosome moves along mRNA until first AUG is reached

• AUG is usually located in Kozak sequence (PuNNAUGG) different from ribosome binding site (5’ Cap)

(ribosome can only bind to 5’ cap)




Shunting Model- first AUG is masked in secondary RNA structure or

- first AUG is not in Kozak sequence

ribosome continues to next AUG


Codon AUG




Multiple translation start sitesinternal ribosome entry site (IRES) is used for ribosomeassembly : consensus site; several hundred nucleotides

multiple open reading frames can be translated from 1 mRNA


Codon AUG



80S

60S

40S

Met

P A

• A (aminoacyl) site: where incoming charged

tRNA binds ribosome (and reads codon inmRNA)

• P (peptidyl) site: where tRNA with growing

polypeptide chain is positioned

Sites on Eukaryotic Ribosome

Modified from Hyde, Introduction to Genetic Principles

P : peptidyl tRNA site

A : aminoacyl tRNA site



Translation : Elongation

Binding the charged tRNA at the A site

• Elongation Factors EF-Tu and EF-Ts• GTP

• Charged tRNAs

• Codon of mRNA positioned in A site ofribosome

E. coli :

Binding a Charged tRNA at the A Site




(E. coli )

Charged tRNA

2. EF-Tu-GTP positions

charged tRNA by binding A siteof ribosome if codon binding is correct

1. Charged tRNA binds EF-Tu-GTP

3.



Translation: ElongationPeptide Bond Formation

• Peptidyl transferase is in the ribosomal RNA of largesubunit of the ribosome (here RNA is the enzyme ; peptidyltransferase is a r ibozyme )

- catalyzes :

1) cleavage of high-energy bond between amino acid

and tRNA in the P site

2) formation of a peptide bond between the a.a. attached

to tRNA in the A site and the a.a attached to tRNA in

the P site



Translation: Elongation

T l ti El ti



Translation: Elongation

Ribosome Translocation :

1. Polypeptide on tRNA inP site is transferred toa.a. on charged tRNA in

A site

2. EF-G enters at A site ;hydrolyzes GTP

3. Ribosome moves down

mRNA one codon4. Uncharged tRNA in P

site moves to E site and

exits ribosome

Cycle of peptide bond formation




and translocation on ribosome





and translocation on ribosome

(1)





(Eukaryotes)

• eEF1 instead of EF-Tu

• eEF1 instead of EF-Ts• eEF2 instead of EF-G

• no E site on ribosome

very similar to E.coli, except :

80S

60S

40S

Met

P A





(Eukaryotes)

eEF1 -GTP positions

charged tRNA by binding A siteof ribosome

eEF1 eEF1

eEF1

eEF1

eEF1

eEF1

eEF1

no E site in ribosome

1. Charged tRNA binds eEF1 -GTP




Error rate : 1 in 10,000 amino acids incorporated into protein

Speed of a.a. incorporation : 15 a.a. per second (E. coli ) 300 a.a. protein made in 20 s !

2-5 a.a. per second (eukaryotes)

300 a.a. protein in 1-2.5 min !





Translation: Termination (E. coli )

Insert Fig.

11.29molecular mimicry : a protein

resembles the shape of ananticodon in a tRNA

Translation: Termination



Translation: Termination

(eukaryotes)

very similar to E.coli except :

• eRF1 instead of RF1 or RF2

• eRF3 similar to RF3

involved with peptide release, GTP hydrolysis

needed

E coli translation is inhibited by



E. coli translation is inhibited by

antibiotics

• azithromycin, erythromycin

block peptide exit tunnel ; prevent elongation

• streptomycin bind to A or P sites and induce errors in

bacterial translation

• tetracycline

block binding of charged tRNA to A site

E coli translation is inhibited by



E. coli translation is inhibited by

antibiotics

30S subunit 50S subunit

Poehlsgaard and Douthwaite, Nature Reviews : Microbiology 3:870-881 (2005)



Energy use in translation

4 high energy phosphate bonds are used per peptide

bond

• 2 high energy phosphate bonds to charge tRNA(tRNA synthetase, ATP AMP)

• 1 high energy phosphate bond to bind the A site(EF-Tu or eEF1 , GTP GDP)

• 1 high energy phosphate bond to translocate (EF-Gor eEF2, GTP GDP)

~90% of bacterial energy production goes into

protein synthesis !

Comparisons : bacterial and



Comparisons : bacterial and

eukaryotic translation

• Prokaryotic mRNA can contain more than one gene per

transcript : polycistronic

• Eukaryotic mRNA contains one gene, monocistronic

- ribosome binds initiation factors recognizing the 5’ cap

Transcription and Translation Are



Transcription and Translation Are

Coupled in E. coli

• eukaryotes : transcription and translation are uncoupled

- transcription : nucleus

- translation : cytoplasm

Polysomes: Many Ribosomes



Polysomes: Many Ribosomes

Bound to mRNA

3’

5’

occur in prokaryotes and eukaryotes

Functional Sites on a Ribosome



Functional Sites on a Ribosome

Growing

polypeptide

mRNA

EF-Tu site

EF-G site

Peptidyl

transferase site

What happens to a

polypeptide once it is

translated on a ribosome ?

- depends on location of

ribosome :

1) free in cytoplasm

2) associated with ER

Targeting mRNA to the



Targeting mRNA to the

Endoplasmic Reticulum

1. Signal peptide: a positively charged a.a.followed by 10-15 hydrophobic amino acids

2. Bound by Signal Recognition Particle (SRP),

halts translation (SRP : 6 proteins and a 7S RNA)

3. SRP binds docking protein on endoplasmic

reticulum

4. Protein translation resumes through

translocation channel (translocon) in ER

5. Signal peptide is removed by signal

peptidase in endoplasmic reticulum

Targeting proteins to ER



Targeting proteins to ER

protein is either secreted from cell or inserted into the membrane

T ti b t i t ER



Targeting membrane proteins to ER

http://kc.njnu.edu.cn/swxbx/shuangyu/4.htm

Transporting membrane proteins to cell surface



Transporting membrane proteins to cell surface

http://kc.njnu.edu.cn/swxbx/shuangyu/4.htm

P tt l ti l Ch t P t i



Posttranslational Changes to Proteins

1. Protein folding

- some proteins spontaneously fold into correct tertiary

structure

- others require chaperones that facilitate proper

folding2. Cleavage of amino terminus : secreted or membrane

proteins

3. Phosphorylation of polypeptide : consensus sequence

4. Addition of sugars or carbohydrates to some R groups

5. Lipid addition

- allows membrane attachment without a signal sequence

* posttranslational modifications : more common in eukaryotes

Ch



Chaperones

- unfold partially folded

protein, allow it to fold in a

different way

- specific to different classes

of proteins

- GroE

GroEL (Hsp60)

GroES (Hsp10)

- Hsp90

- Hsp70




http://www.sciencedirect.com/science/article/pii/S0959437X09000756

http://www.piercenet.com/

http://www.ideacenter.org/contentmgr/showdetails.php/id/838

Phosphorylation

Lipid Addition

Carbohydrate Addition


G ti C d



Genetic Code

• Codons are 3 nucleotide units

- smallest number that can code for 20 a.a.

• Nonoverlapping

• No punctutation (no separation between codons)

• Redundant/degenerate

- more than one codon for some amino acids

• Elucidated using artificially synthesized RNAs (i.e.,

poly U) or trinucleotides – Added to tube containing cell free system

– Asked: What amino acids were joined together in this cell

free system using these RNA templates?

Codons Are Read Three Bases at a Time



Codons Are Read Three Bases at a Time

• Insertion of one

base shifts reading

frame

• Insertion of

two more bases

restores correctreading frame

- insertion or deletion of multiples of 3 nucleotides restored

wild-type function of mutated protein

3 nucleot ides code for 1 am ino acid

Francis Crick :

- mutated viral protein with proflavin : adds or deletes 1 nucleotide

Genetic Code Is Nonoverlapping



Genetic Code Is Nonoverlapping

• position of a.a. next

to each other in

protein indicated

nonover lapping

structure

• no pun ctuat ion

(noncoding nucleotides

between codons)

The Genetic Code



The Genetic Code

Crick, Nirenberg, Matthaei, Leder :

1. mutant phage

2. synthetic mRNAs in cell-free system

- polynucleotide repeats

- dinucleotide repeats

3. synthetic codons

- filter binding assay

tRNA Anticodon Base Pairing with



gmRNA Codon

mRNA and tRNA strands are antiparallel

tRNA

mRNA

“Wobble” in tRNA/mRNA Base



Pairing

• 64 possible codons (3 are stop codons)

• only 50 tRNAs in E. coli

* 3rd base of codon in mRNA and 1st base ofanticodon in tRNA can “wobble”

– base pairing does not have to be exact at this

position

Allows different codons (which differ only in

third position) to be read by same tRNA

Base Pairing Possibilities at Wobble Position



Base Pairing Possibilities at Wobble Position

Insert Fig.

11.45

Universality of Genetic Code



Universality of Genetic Code

• Shared by most living organisms• Some codons are read differently in

– Yeast mitochondrial genes

– Drosophila

– Higher plants

– Mycoplasmas

– Ciliated protozoa

– Site specific variation : interpretation ofcodon depends on its location

E. coli : GUG and UUG sometimes used as

initiator methionine

Design of Genetic Code



Design of Genetic Code

* genetic code is designed to minimize

impact of mutations

– mutations in 3rd position of codon are not likely

to change encoded amino acid- ACU, ACC, ACA, ACG all code for Thr

– functionally related amino acids are encoded bysimilar codons

- all codons with U as middle base encodehydrophobic amino acids (Phe, Leu, Ile, Val)

genetic code is not random

Genetics Chapter 11

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