BiochemistrySixth Edition
Chapter 30Protein Synthesis
Part I
Copyright © 2007 by W. H. Freeman and Company
Berg • Tymoczko • Stryer
Translation: one language to another more complex than replication & txn
1. Many steps and many proteins2. Must be fast (20 amino acid/sec)3. Must be accurate
speed vs. accuracy
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
alanyl-tRNA:First nucleic acid to be sequenced
76 ribonucleotides
Anticodon on alanyl-tRNAis complementary to one of the codons for alanine
General structure of tRNA:
* Cloverleaf* Half of residues are base-paired* Many common structural features why?
General structure of tRNA:
1. 73-93 ribonucleotides2. Enzyme-modified bases
* prevent base pairs
* create hydrophobicity* allow protein interxn
* allow codon recognition
General structure of tRNA:
3. Half of nucleotidesform ds helices5 places without ds:
*acceptor stem*TψC loop*extra arm*DHU loop*anticodon loop
Different butstructurally similar
General structure of tRNA:
4. 5’ pG5. Activated amino acid
attached to 3’ A-OH6. Anticodon (near
center of seq.)
3D structure of yeast phenyl-alanyl-tRNA
3D structure of yeast phenyl-alanyl-tRNA
Important properties:1. L-shaped2. 4 helices 2 ds3. H-bond interactions for nonhelical regions4. 3-terminus: flexible ss5. Anticodon loop: exposed at other end
A good adaptor
Linkage of amino acid to tRNA is crucial:
1. Amino acid-tRNA establishes genetic code2. Activate amino acid for later peptide bond
formation (peptide bond formation: unfavorable)
Activated intermediates:Amino acid esterscarboxyl 2’ or 3’OHof A (tRNA)
Aminoacyl-tRNA orcharged tRNA
Activation reaction of an amino acid
Catalyzed by specific aminoacyl-tRNA synthetases (or activating enzymes)
Amino acid + ATP amionacyl-AMP + PPi
Or amionacyl-AMP
The second step of the reaction
amionacyl-AMP + tRNAaminoacyl-tRNA + AMP
Sum of reaction
Amino acid + ATP + tRNA + H2Oaminoacyl-tRNA + AMP + 2Pi
* 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
How does aminoacyl-tRNA synthetase incoporate the correct amino acid?
1. Specific structure of theamino acid binding site
zinc ion + Asp
other synthetase havedifferent active sitestructures
2. 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)
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 largeramino acids
Editing site cleaves activated amino acids thatare smaller than the correct one
Editing mechanism:
The flexible CCA arm can swing out of the activation site and into the editing site
Editing without dissociating (fidelity )
Editing mechanism:
High accuracy can stillbe achieved without editing
Proofreading: initial a.a.binding interaction is not good enough
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
How do synthetases choose their tRNA partners?
1. Anticodon?
Some synthetases recognize their tRNA partnersprimarily based on the anticodon loop.
Threonyl-tRNA synthetase& tRNAThr
5’-CGU-3’ H bonds with G and U
How do synthetases choose their tRNA partners?
2. microhelix in tRNA
A 24 nt microhelix canbe aminoacylated by alanyl-tRNA synthetase(without anticodon)
Mutated tRNACys
can be recognized bysynthetase (alanine)
Aminoacyl-tRNA synthetases: diverse (independent evolution?)
Structural and sequence comparisons They are related
Synthetases fall into two classes!
Differences between the two classes:
1. Different binding surfaces2. CAA arm conformations3. OH group acylation4. ATP-binding conformations5. Monomeric vs. dimeric
Ribosome: ribonucleoprotein particle with large and small subunits
L1-L3423S & 5S rRNA
S1-S2116S rRNA
Ribosome:RNA is 2/3 of total mass
30S primary transcript 5S, 16S, 23S
Extensive foldingInternal base pairs(conserved base-pairs,not conserved seq.ex. G-C vs. A-U)
16S rRNA 2o structure* Defined structure* Short duplex
“ Chicken and egg” question:If ribosomes synthesize proteins, where doribosomal protiens come from?
Protein: catalysisRNA: structural
OR
RNA: catalysisProtein: structural and regulation
Proteins are synthesized in the N to C direction
How is mRNA read?
The direction of translation is 5’ 3’
The direction of txnis also 5’ to 3’ Same direction
So there is a couplingbetween txn and tsl Efficiency
Polyribosome or polysome
Translation initiation The first codon is more than 25nt from 5’
Ribonuclease digestion: initiator region on mRNA
12
3 ~ 9 bp
Two kinds of interactions determine tsl initiation
Translation is initiated by
formylmethionyl-tRNAf
tRNAf vs. tRNAm
Ribosomes have 3 tRNA-binding sites
ExitPeptidylAminoacyl
Tunnel for protein escape
Mechanism of protein synthesis
1. 30S + mRNA
2. tRNAf Met
3. 50S
Peptidyl transferase center on 23S rRNA
Center: promote reaction & stabilize intermediate
The peptide chain remains in the P site on the 50S (tunnel)
The amino acid in the aminoacyl-tRNA does not play a role in selecting a codon
Codon-anticodon interaction:
* Watson-crick base pairing* Anti-parallel* One anticodon for one codon?
Some tRNA recognize morethan one codon:Alanyl-tRNA: GCU, GCC, GCA
Degeneracy of genetic code:XYU & XYCXYA & XYG
Wobble: steric freedom
Appears in several anticodon
Anticodon of yeast alanyl-tRNA: IGCCodons: GCU, GCC, GCA
Codon-anticodon interactions:
1. 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)
2. First base of anticodon determines how many codons to be read degeneracy of genetic code: from wobble interxn
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