Chapter 11

Gene Expression: Translation

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

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

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

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

dipolar ion (zwitterion)

Amino acids are joined together by peptide bonds dehydration synthesis

Protein Structure
Primary: Linear sequence of amino acids Secondary: Common folding patterns that form due to 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 one polypeptide to form active protein
** each level of structure depends on the level below it

Secondary Structure

-helix

-pleated sheet

Tertiary Structure
Three-dimensional structure of human -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. coli ribosome A : aminoacyl site P : peptidyl site E : exit site

Aminoacyl-tRNA synthetases
Covalently attaches (charges) the correct amino acid to 3 end of the correct tRNA : recognizes acceptor stem and anticodon 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 more bases 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

IF1 binds at A site, prevents 1st tRNA from binding there
(N-formyl-methionine)

IF2 only binds fMet fMet is the only initiator aa

binds at P site of 30S

E : exit site P : peptidyl tRNA site A : aminoacyl tRNA site

Initiation of Translation in E.coli : Shine-Dalgarno sequence in mRNA base pairs with16S rRNA
mRNA

(just upstream of initiator AUG)

16S rRNA

Initiation of Translation in Eukaryotes

Preinitiation complex requires: Small subunit (40S) of the ribosome 5 cap mRNA Initiation factors eIF2, eIF3, eIF4 Met Initiator Met-tRNA i GTP

Initiation of Translation in Eukaryotes

Initiator complex forms at 5 cap : eIF3 binds 40S subunit eIF2-GTP binds Met-tRNA and then binds 40S subunit
Met i
GTP

eIF4 binds 5 Cap of mRNA 5 Cap of mRNA binds complex of 40S subunit, eIF2-GTP, eIF3, and Met-tRNAMet i

GTP

GTP

PABP interactions with initiator complexes enhance translation by helping initiation and stabilizing mRNA
polyA binding protein

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

may also insure that only mature mRNAs are translated

Initiation of Translation in Eukaryotes: Finding the Start Codon (AUG)

binding of eIF1 and eIF1A stimulates 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-tRNAMet to small i ribosomal subunit
eIF2B required to reconstitute the active form of eIF2 (eIF2-GTP) from eIF2-GDP

Mechanisms to Identify Start Codon AUG

AUG is usually located in Kozak sequence (PuNNAUGG) different from ribosome binding site (5 Cap) Scanning Model ribosome moves along mRNA until first AUG is reached

(ribosome can only bind to 5 cap)

Mechanisms to Identify Start Codon AUG

Shunting Model - first AUG is masked in secondary RNA structure or - first AUG is not in Kozak sequence ribosome continues to next AUG

Mechanisms to Identify Start Codon AUG

Multiple translation start sites internal ribosome entry site (IRES) is used for ribosome assembly : consensus site; several hundred nucleotides multiple open reading frames can be translated from 1 mRNA

Sites on Eukaryotic Ribosome

A (aminoacyl) site: where incoming charged tRNA binds ribosome (and reads codon in mRNA) P (peptidyl) site: where tRNA with growing polypeptide chain is positioned
60S Met P : peptidyl tRNA site A : aminoacyl tRNA site

80S

A
Modified from Hyde, Introduction to Genetic Principles

40S

Translation : Elongation
Binding the charged tRNA at the A site E. coli : Elongation Factors EF-Tu and EF-Ts GTP Charged tRNAs Codon of mRNA positioned in A site of ribosome

Binding a Charged tRNA at the A Site (E. coli)

1. Charged tRNA binds EF-Tu-GTP

Charged tRNA

3.

2. EF-Tu-GTP positions charged tRNA by binding A site of ribosome if codon binding is correct

Translation: Elongation
Peptide Bond Formation Peptidyl transferase is in the ribosomal RNA of large subunit of the ribosome (here RNA is the enzyme ; peptidyl transferase is a ribozyme) - 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

Translation: Elongation
Ribosome Translocation : 1. Polypeptide on tRNA in P site is transferred to a.a. on charged tRNA in A site 2. EF-G enters at A site ; hydrolyzes GTP 3. Ribosome moves down mRNA one codon 4. Uncharged tRNA in P site moves to E site and exits ribosome

Cycle of peptide bond formation and translocation on ribosome

Cycle of peptide bond formation and translocation on ribosome

(1)

Translation : Elongation (Eukaryotes)

very similar to E.coli, except : eEF1 instead of EF-Tu eEF1 instead of EF-Ts eEF2 instead of EF-G no E site on ribosome
Met 60S

80S
P A

40S

Binding a Charged tRNA at the A Site (Eukaryotes)

1. Charged tRNA binds eEF1 -GTP
eEF1 eEF1

eEF1 eEF1 eEF1

eEF1

eEF1

no E site in ribosome

eEF1 -GTP positions charged tRNA by binding A site of ribosome

Translation : Elongation
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)

Release factor (RF1 or RF2) binds STOP codon in A site (STOP codon : UAA, UGA, UAG) carboxyl end of polypeptide is hydrolyzed from tRNA in P site release factor (RF3) hydrolyzes GTP to release RF1 or RF2 Ribosomal recycling factor (RRF) binds A site and is translocated to the P site with help of EF-G initiation factors help to dissociate and recycle ribosomal subunits for next round

Translation: Termination (E. coli)

Insert Fig. 11.29

molecular mimicry : a protein resembles the shape of an anticodon in a tRNA

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 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 antibiotics

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

30S subunit

50S subunit

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-G or eEF2, GTP GDP)

~90% of bacterial energy production goes into protein synthesis !

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 Coupled in E. coli

eukaryotes : transcription and translation are uncoupled - transcription : nucleus - translation : cytoplasm

Polysomes: Many Ribosomes Bound to mRNA

5
occur in prokaryotes and eukaryotes

Functional Sites on a Ribosome

Peptidyl transferase site EF-G site EF-Tu 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

mRNA

Growing polypeptide

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

protein is either secreted from cell or inserted into the membrane

Targeting membrane proteins to ER

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

Transporting membrane proteins to cell surface

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

Posttranslational Changes to Proteins

1. Protein folding - some proteins spontaneously fold into correct tertiary structure - others require chaperones that facilitate proper folding 2. 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

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

Posttranslational Changes to Proteins

Phosphorylation Lipid Addition

Carbohydrate Addition

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

http://www.piercenet.com/

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

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

Francis Crick : - mutated viral protein with proflavin : adds or deletes 1 nucleotide Insertion of one base shifts reading frame

Insertion of two more bases restores correct reading frame - insertion or deletion of multiples of 3 nucleotides restored wild-type function of mutated protein 3 nucleotides code for 1 amino acid

Genetic Code Is Nonoverlapping

position of a.a. next to each other in protein indicated nonoverlapping structure

no punctuation (noncoding nucleotides between codons)

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 mRNA 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 of anticodon 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

Insert Fig. 11.45

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 of codon depends on its location E. coli : GUG and UUG sometimes used as initiator methionine

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 by similar codons - all codons with U as middle base encode hydrophobic amino acids (Phe, Leu, Ile, Val) genetic code is not random