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