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

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 .Genes control enzymes : Beadle and Tatum Isolated mutant strains of Neurospora that could only grow if niacin was supplied in culture medium (auxotrophs) .

may result in multiple symptoms (phenotypes): ulcers in corneas. lesion on skin. mental retardation .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.

Basic 3. Acidic 2. Nonpolar (hydrophobic) 4. Polar (uncharged) . four classes: 1.Proteins Are Composed of Amino Acids • Amino (NH2) group • Carboxyl (COOH) group • Side chains (R groups).

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” .

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 .Protein Structure • Primary: Linear sequence of amino acids • Secondary: Common folding patterns that form due to maximization of hydrogen bonds (alpha helix.

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 .

amino acid reacts with ATP (binds AMP. releases PPi) 2. amino acid is detached from AMP and joined to 3’ end of the tRNA .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.

3 for STOP codons) anticodon of tRNA. and not the amino acid itself.a. is recognized and dictates which amino acid is incorporated anticodon anticodon .Decoding Machinery • • • • 20 different aminoacyl tRNA synthetases More than 20 tRNAs 64 codons (61 code for a. determines which amino acid is incorporated result : Ala incorporated into protein  anticodon on tRNA. . not amino acid.

positioning 1st charged tRNA at P site • Elongation .recognition of 1st codon .Translation • Initiation .recognize STOP codon .stop elongation .a.add a.release of polypeptide . from charged tRNAs at A site • Termination .formation of ribosome .

resulting in a nonfunctional protein . resulting in shift of reading frame .Translational Initiation Must Start at Correct Codon • Frameshift mutations: deletion or insertion of one or more bases in mRNA.If ribosome shifts out of frame it translates different codons.

Initiator methionine in E. coli (initiator methionine) (internal methionine) .

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.

Initiation of Translation in E.coli : Shine-Dalgarno sequence in mRNA base pairs with16S rRNA mRNA (just upstream of initiator AUG) 16S rRNA .

eIF4 Met • Initiator Met-tRNA i • GTP .Initiation of Translation in Eukaryotes Preinitiation complex requires: • Small subunit (40S) of the ribosome • 5’ cap mRNA • Initiation factors eIF2. eIF3.

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. and Met-tRNAMet i GTP GTP . eIF2-GTP. eIF3.

com/horizon/rna/highlights/figures/s2_spec1_f1.PABP interactions with initiator complexes enhance translation by helping initiation and stabilizing mRNA polyA binding protein http://www.html  may also insure that only mature mRNAs are translated .nature.

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 not in Kozak sequence  ribosome continues to next AUG .first AUG is masked in secondary RNA structure or .

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 .

Introduction to Genetic Principles 40S .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 P A Modified from Hyde.

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

EF-Tu-GTP positions charged tRNA by binding A site of ribosome if codon binding is correct . Charged tRNA binds EF-Tu-GTP Charged tRNA 3. coli) 1.Binding a Charged tRNA at the A Site (E. 2.

a. attached to tRNA in the A site and the a.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.Translation: Elongation Peptide Bond Formation • Peptidyl transferase is in the ribosomal RNA of large subunit of the ribosome (here RNA is the enzyme .a attached to tRNA in the P site . peptidyl transferase is a ribozyme) .

Translation: Elongation .

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

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 .

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 .Binding a Charged tRNA at the A Site (Eukaryotes) 1.

Translation : Elongation Error rate : 1 in 10.5 min ! . incorporation : 15 a.a.a. protein in 1-2.a. coli)  300 a. protein made in 20 s ! 2-5 a.000 amino acids incorporated into protein Speed of a.a. per second (eukaryotes)  300 a.a. per second (E.

Translation: Termination (E. UGA.coli) • Release factor (RF1 or RF2) binds STOP codon in A site (STOP codon : UAA. 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 .

29 molecular mimicry : a protein resembles the shape of an anticodon in a tRNA . 11.Translation: Termination (E. coli) Insert Fig.

GTP hydrolysis needed .Translation: Termination (eukaryotes) very similar to E.coli except : • eRF1 instead of RF1 or RF2 • eRF3 similar to RF3  involved with peptide release.

erythromycin  block peptide exit tunnel . coli translation is inhibited by antibiotics • azithromycin. 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.

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. GTP  GDP)  ~90% of bacterial energy production goes into protein synthesis ! . 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.

ribosome binds initiation factors recognizing the 5’ cap . monocistronic .Comparisons : bacterial and eukaryotic translation • Prokaryotic mRNA can contain more than one gene per transcript : polycistronic • Eukaryotic mRNA contains one gene.

Transcription and Translation Are Coupled in E.translation : cytoplasm .transcription : nucleus . coli • eukaryotes : transcription and translation are uncoupled .

Polysomes: Many Ribosomes Bound to mRNA 3’ 5’  occur in prokaryotes and eukaryotes .

depends on location of ribosome : 1) free in cytoplasm 2) associated with ER mRNA Growing polypeptide .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 ? .

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

htm .Transporting membrane proteins to cell surface http://kc.njnu.cn/swxbx/shuangyu/4.edu.

Posttranslational Changes to Proteins 1.some proteins spontaneously fold into correct tertiary structure .others require chaperones that facilitate proper folding 2.allows membrane attachment without a signal sequence * posttranslational modifications : more common in eukaryotes . Addition of sugars or carbohydrates to some R groups 5. Protein folding . Cleavage of amino terminus : secreted or membrane proteins 3. Phosphorylation of polypeptide : consensus sequence 4. Lipid addition .

Hsp90 .Chaperones .specific to different classes of proteins .GroE GroEL (Hsp60) GroES (Hsp10) .unfold partially folded protein. allow it to fold in a different way .Hsp70 .

Posttranslational Changes to Proteins Phosphorylation Lipid Addition Carbohydrate Addition http://www.org/contentmgr/showdetails.php/id/838 .ideacenter.com/ http://www.sciencedirect.com/science/article/pii/S0959437X09000756 http://www.piercenet.

Genetic Code • Codons are 3 nucleotide units . 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? ..e.more than one codon for some amino acids • Elucidated using artificially synthesized RNAs (i. • Nonoverlapping • No punctutation (no separation between codons) • Redundant/degenerate .smallest number that can code for 20 a.a.

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 .Codons Are Read Three Bases at a Time Francis Crick : .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) .

Leder : 1.filter binding assay . Matthaei. mutant phage 2.polynucleotide repeats . synthetic mRNAs in cell-free system . Nirenberg.dinucleotide repeats 3.The Genetic Code Crick. synthetic codons .

tRNA Anticodon Base Pairing with mRNA Codon mRNA and tRNA strands are antiparallel tRNA mRNA .

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 .“Wobble” in tRNA/mRNA Base Pairing • 64 possible codons (3 are stop codons) • only 50 tRNAs in E.

45 .Base Pairing Possibilities at Wobble Position Insert Fig. 11.

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 .

ACG all code for Thr – functionally related amino acids are encoded by similar codons . Ile. ACC. Leu.all codons with U as middle base encode hydrophobic amino acids (Phe.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 . Val)  genetic code is not random .ACU. ACA.

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