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Translation Chapter 9 Overview Occurs on ribosomes-large aggregates of rRNA and protein tRNA acts as amino acid carriers Prokaryotes—occurs simultaneously with transcription and mRNA degradation Eukaryotes—occurs in cytoplasm mRNA translated 5’3’ Protein synthesis aminocarboxy Protein Synthesis Polymerization of amino acids: condensation reaction (dehydration synthesis) ~Universal Genetic Code Codons—sets of 3 nucleotides corresponding to a single amino acid Each codon specifies a single amino acid More than one codon can specify the same amino acid code is said to be degenerate Some aa correspond to a single codon AUG—initiator codon, methionine (Met, M) UGG–Tryoptophan (TrP, W) Often codons encoding the same aa differ onl;y at the 3rd nucleotide ~Universal Genetic Code Why~Universal? Exceptions GUG sometimes used as a start Mammalian mitochondria NH Ciliated protozoa Selenocysteine H-C-CH Se 3 + 2 COO- Selenocysteine The 21st amino acid? An essential amino acid for selenoproteins EX. Glutathione oxidase Uses unique tRNA (tRNASec), initially bound to Ser. Longest known tRNA (95nt). Anticodon recognizes UGA (Stop) as Sec Signal (a stem loop configuration) 3’ to the UGA determine Stop or Sec Dedicated specific elongation factor recognizes the stem-loop and substitutes for usual elongation factor (EF-Tu) Degeneracy—Wobble Hypothesis Explains how some tRNA recognize more than one codons tRNA molecules only need to make strong base pairs with 2 of the three codons in the nucleotide This third loose base pairing interaction is called wobble Note: only certain bases can substitute for others Wobble Example This UCA codon was read by the tRNA with a UGA anticodon But if this UCA was UCG, it would still have been read by the tRNA with a UGA anticodon Codon Usage More than one codon exists for most amino acids (except Met and Trp) Organism may have a preferred codon for a particular amino acid Codon usage correlates with abundance of tRNAs (preferred codons are represented by abundant tRNAs) Rare tRNAs correspond to rarely used codons mRNAs containing rare codons experience slow translation Implications for GE? Amino Acyl Synthetase and tRNA Amino acyl synthetases catalyze attachment of aa to its appropriate tRNA One for each amino acid tRNA Derived from large 1º transcript Heavily modified, unusual bases Extensive folding due to internal H-bonding Amino Acyl Synthetase Carboxy end of aa attached to -phospate of ATP AMP released as carboxy end of amoinoacyl group transferred to O at C-3 of 3’nt When aa is attached, tRNA is charged or acylated No aa = uncharged Wrong aa = mischarged NOTE PPi tRNA Activation by Aminoacyl tRNA Synthetases O 1. Aminoacyl-AMP formation: HO (-)O O P R +H3N O O(-) P O R O(-) +H3N O C P O O- Adenine O O C O O P O O Adenine O O- OH OH Aminoacyl adenylate (Aminoacyl-AMP) OH OH + PPi 2Pi 2. Aminoacyl transfer to the appropriate tRNA: R +H3N R C O O O P O O- Adenine O + HO-ACC-tRNA +H3N C O ACC-tRNA + AMP O OH OH Overall reaction: amino acid + tRNA + ATP aminoacyl-tRNA + AMP + PPi tRNA Function and Structure Anticodoncomplementary to codon on mRNA Amino attachment (CCA) site Other recognition sites DHU loop TC Loop Extra arm (variable) NOTE: also unusual bases observed acceptor stem acceptor stem tRNA Recognition by Amino Acyl Synthetase Sequence elements in each tRNA are recognized by its specific synthetase including: One or more bases in acceptor stem Base at position 73 “Discriminator base” Seems to play a major role in many cases, but in other cases it is completely ignored. In many, at least one anticodon base Recognition (cont’d) No common set of rules for tRNA recognition !!! Anticodon region is not the only recognition site The "inside of the L" and other regions of the tRNA molecule are also important Specificity of several aminoacyl-tRNA synthetases determined by: one or more bases in anticodon one or more bases in the acceptor stem discriminator base 73 Mischarging Observation: several aa similar in size and shape, but mischarging rare. Editing carried out by aminoacyl tRNA synthetase Ex Double sieve of isoleucine synthetase Activation site– coarse sieve, rejects aa larger than ile. excluded because they don’t fit. Editing (hydrolytic) site—fine sieve. Accepts activated amino acids that are smaller than ile (ex, Val-AMP), but rejects Ile-AMP (too large). those that get through are hydrolyzed to aa and AMP. Reduces mischarging from 1/225 (expected) to 1/180,000 (observed). Sites can also distinguish based on hydrophobicity Isoleucil-tRNA Synthetase: Proofreading Based on Size Larger Acylation Site Larger Acylation Site Smaller Hydrolytic Site Smaller Hydrolytic Site CH 3 H3C CH 3 CH 3 O O NH 3 + +H 3 N tRNAIle O CH 3 O tRNAIle Difference in Size H 3C CH 3 O O +H 3 N +H 3 N O CH 3 O tRNAIle Ile Correct Acylation Val Misacylation tRNAIle Valyl tRNAVal Synthetase Proofreading: Hydrophobic/Polar Recognition Motif Hydrophobic Acylation Site 3 HC Polar Hydrolytic Site Hydrophobic Acylation Site Polar Hydrolytic Site CH 3 H 3C O OH O +H 3 N NH 3 + O tRNAVal tRNAVal O Difference in Hydrophobicity CH3 CH 3 HO CH 3 O O +H 3 N +H 3 N O tRNAVal Val Correct Acylation O tRNAVal Thr Misacylation Experiment (1962) tRNA-ACA Cell-free extract amino acids & enymes tRNA is charged with Cys Cys-tRNA-ACA Treat w metal catalyst removes thiol groups Anticodon (recognizes UGU codon, encodes Cys) RNA template UGUGUGUGUG... Protein has Cys Charged amino acid is changed chemically Ala-tRNA-ACA RNA template UGUGUGUGUG... Protein has Ala Once an aminoacyl-tRNA has been synthesized the amino acid part makes no contribution to accurate translation of the mRNA. Protein Synthesis-3 Stages Initiation Elongation Termination Ribosomes Composition of eukaryotic and prokaryotic ribosomes Mol. Biol. Gene, Fig. 14-13 Composition of the E. coli Ribosome 50S subunit 23S & 5S RNA + 34 proteins 30S subunit 16S RNA + 21 proteins Gross anatomy of the E. coli ribosome. Fig. 19.5 head platform stalk platform ridge Central protuberance stalk Initiation In both prokaryotes and eukaryotes, protein synthesis begins with a specific initiating tRNA In prokaryotes, initiator methionine amino group is methylated Attached to special tRNA (fMet-tRNA) Transformylase—adds formyl group Deformylase—removes formyl group from Met of completed peptide Formylation does not occur in eukaryotes Steps in Initiation Association of 30S subunit with mRNA fMet-tRNA Initiation factors (3 proteins) GTP } 30S Preinitiation Complex QUESTION: AUG encodes fMet and Met. How does 30S ribosome “know” which aa is to be inserted? Initiator Codon Recognition fMet-tRNA responds only to initiator codons (AUG, GUG, UUG [rarely]) Met-tRNA responds only to internal AUG Meaning of codons dependent on their context, i.e., sequences nearby In Eukaryotes: 5’ cap involvement In Prokaryotes: Shine-Dalgarno Sequence mRNA- (5’)AGGAG (3’) 16S rRNA- (3’)UCCUC(5’) Shine-Dalgarno Interaction Upstream from initiator AUG Complement ary to a stretch on 16S rRNA Seen in virtually all prokaryotic mRNA Initiation Factors in Protein Synthesis IF-1 Promotes dissociation of ribosome. IF-1 also blocks the A site of the small ribosomal subunit insures the initiation aa-tRNA fMet-tRNAfMet can bind only in the P site & that no other aatRNA can bind in the A site during initiation. IF-2 small GTP-binding protein (a GTPase). Interacts with Small subunit IF1 fMet-tRNAfMet Initiation Factors in Protein Synthesis IF-2 (cont’d) IF-2/GTP helps the initiator helps it dock with the small ribosome subunit, prevents other tRNAs from binding small subunit IF-3 Binds small subunit, prevents reassociation w/ large subunit binds mRNA to the 30S ribosomal subunit frees it from its complex with the 50S subunit. 30S Pre-initiation Complex 70S Initiation Complex Assembly 30S pre-initiation= 30S subunit,IF1-3, mRNA, GTP, fMettRNAfMet When fMet-tRNAfMet pairs with initiator codon, small subunit undergoes conformational change Result: Release of IF-3 Binding of large subunit stimulates GTPAse activity of IF2/GTP Large subunit can bind small subunit complex hydrolysis of GTP to GDP IF-2/GDP and IF-1 fall off RESULT: 70S ribosome with fMet-tRNAfMet in P-site of ribosome RESULT: 70S Initiation Complex Overview Dissociation of inactive 70S IF-1, IF-3 IF-2/tRNA, mRNA fMet-tRNAfMet/initiator codon-releases IF-3 GTP Hydrolysis, releases IF-1, IF-2 Complete 70S complex 70S Ribosome A (aminoacyl) site Small subunit Transfer RNAs P (peptidyl) site Large subunit (50 S) 5’ end Messenger RNA Alternative View A=Aminoacyl site P=peptidyl site E=Exit site Elongation Overview Aminoacyl tRNA complementary to codon in Asite moves into A-site N-formyl-Met transferred from tRNAfMet to aminoacyl-tRNA in A-site tRNAfMet leaves P-site Ribosome moves along mRNA (translocation) now have a dipeptide in the A-site dipeptide in P-site New aminoacyl-tRNA moves into A site, etc Elongation ELONGATION Transpeptidation Reaction tRNA P site O O O P O A site tRNA O CH2 H Carboxy end of nascent peptide O O O P H O H OH H O C R NH C HC O CH2 O H HC O Adenine R NH3+ O H H O H OH C HC Nucleophilic attack Adenine R :NH2 Amino terminus of incoming amino acid Transpeptidation Completed tRNA The nascent polypeptide, one residue longer, is now linked to the tRNA in the A site. P site O O O P O A site tRNA O CH2 H Adenine O H H OH H OH O P O O CH2 H O O H H O H OH C HC R NH O C HC R NH O Adenine C HC R NH3+ EF-GGTP Role in translocation Binding site uncovered EF-G-GTP occupies Hydrolysis GDP leaves open A-site Translation Termination “Stop” Codon No anticodons, but are release factors Proteins Occupy A-site Activate hydrolysis of peptide from peptidyl-tRNA Translation Termination 2 Release factors RF-1 and RF-2 recognize stop codons RF-3 –stimulates dissociation of 70S ribosome after release of polypeptide chain Anticodon recognition determined 3 aa Proofreading in Translation 1. Codon:anticodon base pairing 2. 16S rRNA forms Hbonds with minor groove of codon:anticodon duplex only when correctly paired 3. Proofreading in Translation Correct base pairing allows EF-Tu bound to aa-tRNA to interact with factor binding center, inducing GTP hydrolysis and EF-Tu release Incorrect base pairing FBC not contacted allows more time for EFTu GTP release 4. Proofreading in Translation Incorrectly paired tRNA can’t rotate into position for peptide bond formation “tRNA accommodation” Antibiotics Translation the target of many antibiotics. EX Site of Nucleophilic attack Absent terminates translation Eukaryotic Translation Factors designated with the prefix "e" EF-Ts replaced by eEF-1 eEF-2 (target for diphtheria toxin) Diphtheria Toxin EF2 is only known substrate for diphtheria toxin Addition of ADP-ribose inactivates EF2 EF2 contains rare modification of one of histidine residues and this is site recognized by toxin Mutant cells that cannot modify site are resistant Kills cells by irreversible block of protein synthesis P. aeruginosa exotoxin A works same as diphtheria toxin