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Berg • Tymoczko • Stryer
Biochemistry Sixth Edition Chapter 30 Protein Synthesis Part I Copyright © 2007 by W. H. Freeman and Company
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Translation: one language to another
more complex than replication & txn Many steps and many proteins Must be fast (20 amino acid/sec) Must be accurate
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speed vs. accuracy
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Fidelity of translation:
correct recognition of codons on mRNA An amino acid itself cannot recognize codon: A transfer is required: tRNA tRNA: adapter molecule that binds to a specific codon and brings an amino acid for incorporation into polypeptide chain
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alanyl-tRNA: First nucleic acid to be sequenced 76 ribonucleotides
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Anticodon on alanyl-tRNA
is complementary to one of the codons for alanine
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General structure of tRNA:
* Cloverleaf * Half of residues are base-paired * Many common structural features why?
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General structure of tRNA: 73-93 ribonucleotides Enzyme-modified bases
* prevent base pairs * create hydrophobicity * allow protein interxn * allow codon recognition
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General structure of tRNA: Half of nucleotides form ds helices
5 places without ds: *acceptor stem *TψC loop *extra arm *DHU loop *anticodon loop Different but structurally similar
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General structure of tRNA:
5’ pG Activated amino acid attached to 3’ A-OH Anticodon (near center of seq.)
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3D structure of yeast phenyl- alanyl-tRNA
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3D structure of yeast phenyl- alanyl-tRNA Important properties: 1. L-shaped 2. 4 helices 2 ds 3. H-bond interactions for nonhelical regions 4. 3-terminus: flexible ss 5. Anticodon loop: exposed at other end A good adaptor
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Linkage of amino acid to tRNA is crucial:
Amino acid-tRNA establishes genetic code Activate amino acid for later peptide bond formation (peptide bond formation: unfavorable)
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Activated intermediates:
Amino acid esters carboxyl 2’ or 3’OH of A (tRNA) Aminoacyl-tRNA or charged tRNA
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Activation reaction of an amino acid
Catalyzed by specific aminoacyl-tRNA synthetases (or activating enzymes) Amino acid + ATP amionacyl-AMP + PPi
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Or amionacyl-AMP
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The second step of the reaction
amionacyl-AMP + tRNA aminoacyl-tRNA + AMP Sum of reaction Amino acid + ATP + tRNA + H2O aminoacyl-tRNA + AMP + 2Pi
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* Equivalent of 2 ATP is consumed in activation
* Activation and transfer steps for a particular amino acid are catalyzed by the same amino- acyl-tRNA synthetase (intermediate does not dissociate from enzyme, stably bound to active site) * Acyl adenylate intermediate (also in fatty acid activation
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How does aminoacyl-tRNA synthetase incoporate
the correct amino acid?
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Specific structure of the
amino acid binding site zinc ion + Asp other synthetase have different active site structures
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Proofreading by amino- acyl-tRNA synthetase
what happens when: threonyl-tRNA synthetase + Ser-tRNAThr serine + tRNA editing: hydrolysis of wrong amino acid (carried out by editing site; size exclusion)
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editing: hydrolysis of wrong amino acid
(carried out by editing site; size exclusion) Most synthetases contain both editing site & activation site Activation (or acylation) site rejects larger amino acids Editing site cleaves activated amino acids that are smaller than the correct one
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Editing mechanism: The flexible CCA arm can
swing out of the activation site and into the editing site Editing without dissociating (fidelity )
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Editing mechanism: High accuracy can still be achieved without editing
Proofreading: initial a.a. binding interaction is not good enough
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How do synthetases choose their tRNA partners?
synthetases are the only molecules that “know” the genetic code! precise recognition of tRNAs recognition is different for each synthetase- tRNA
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How do synthetases choose their tRNA partners?
Anticodon? Some synthetases recognize their tRNA partners primarily based on the anticodon loop.
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Threonyl-tRNA synthetase
& tRNAThr 5’-CGU-3’ H bonds with G and U
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How do synthetases choose their tRNA partners? microhelix in tRNA
A 24 nt microhelix can be aminoacylated by alanyl-tRNA synthetase (without anticodon) Mutated tRNACys can be recognized by synthetase (alanine)
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Aminoacyl-tRNA synthetases:
diverse (independent evolution?) Structural and sequence comparisons They are related Synthetases fall into two classes!
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Differences between the two classes:
Different binding surfaces CAA arm conformations OH group acylation ATP-binding conformations Monomeric vs. dimeric
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Ribosome: ribonucleoprotein particle with
large and small subunits L1-L34 23S & 5S rRNA S1-S21 16S rRNA
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Ribosome: RNA is 2/3 of total mass 30S primary transcript 5S, 16S, 23S
Extensive folding Internal base pairs (conserved base-pairs, not conserved seq. ex. G-C vs. A-U) 16S rRNA 2o structure * Defined structure * Short duplex
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“Chicken and egg” question:
If ribosomes synthesize proteins, where do ribosomal protiens come from? Protein: catalysis RNA: structural OR RNA: catalysis Protein: structural and regulation
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Proteins are synthesized in the N to C direction
How is mRNA read?
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The direction of translation is 5’ 3’
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The direction of txn is also 5’ to 3’ Same direction So there is a coupling between txn and tsl Efficiency Polyribosome or polysome
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Translation initiation
The first codon is more than 25nt from 5’ Ribonuclease digestion: initiator region on mRNA 2 1
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3 ~ 9 bp Two kinds of interactions determine tsl initiation
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Translation is initiated by
formylmethionyl-tRNAf tRNAf vs. tRNAm
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Ribosomes have 3 tRNA-binding sites
Exit Peptidyl Aminoacyl
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Tunnel for protein escape
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Mechanism of protein synthesis
30S + mRNA tRNAf Met 50S
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Peptidyl transferase center on 23S rRNA
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Center: promote reaction & stabilize intermediate
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The peptide chain remains in the P site on the 50S (tunnel)
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The amino acid in the aminoacyl-tRNA does not
play a role in selecting a codon
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Codon-anticodon interaction:
* Watson-crick base pairing * Anti-parallel * One anticodon for one codon? Some tRNA recognize more than one codon: Alanyl-tRNA: GCU, GCC, GCA Degeneracy of genetic code: XYU & XYC XYA & XYG
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Wobble: steric freedom
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Appears in several anticodon
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Anticodon of yeast alanyl-tRNA: IGC
Codons: GCU, GCC, GCA
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Codon-anticodon interactions:
The first two bases of codon: standard pairing codons differ in the first 2 bases are recognized by different tRNAs (ex. UUA and CUA of leucine) First base of anticodon determines how many codons to be read degeneracy of genetic code: from wobble interxn
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Why wobble only in the third base of codon?
30S/16S rRNA: A1492, A1493, G530 forms H bond with first 2 paired anticodon-codon (check WC bp) Ribosome plays an active role in decoding codon- anticodon interactions
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