protein synthesis ii biochemistry 302 - university of...

Protein synthesis IIBiochemistry 302

Bob Kelm February 23, 2005

Several idealized views of the 70S ribosomal complex during translation

Both models imagine all three binding sites (A, P, E) occupied by tRNAs. This would only be a transient occurrence during actual protein synthesis.

50S “tunnel”

70S cavity

Fig. 27.25

View with 30S subunit in front, 50S subunit behind

Prokaryotic translation: Three steps of chain elongation• A site (AA-tRNA binding, EF-Tu-GTP hydrolysis)

– Loading of new AA-tRNA joined to EF-Tu-GTP– Codon positioning of AA-tRNA assisted by GTP hydrolysis– Dissociation of EF-Tu-GDP (Released EF-Tu “reloaded” with

GTP via EF-Ts exchange factor)• A,P sites (transpeptidation in 50S subunit)

– α-amino group from A site AA-tRNA attacks the carbonyl carbon of P-site bound peptidyl-tRNA

– Formation of new peptide bond at A/P 50S hybrid-site– P-site deacylated tRNA (i.e. w/o peptide) - leaving group

• A, P, E site (translocation, EF-G-GTP hydrolysis)– Rapid transfer of uncharged tRNA to E site and ejection – Translocation of peptidyl-(3′OH) tRNA from A site to P site via

EF-G-mediated ribosome movement 3′ to the next codon

Stage 3: Elongation Step 1: Binding of second aminoacyl-tRNA (EF-Tu-GTP)

Proofreading of codon anticodon interaction likely occurs at this step to allow “incorrect” aminoacyl-tRNAsto dissociate.

Lehninger Principles of Biochemistry, 4th ed., Ch 27

Regeneration of EF-Tu-GTP: This “reloading” cycle does not involve any GTP hydrolysis. Regeneration occurs by EF-Ts mediated nucleotide exchange.

Stage 3: Elongation Step 2: Formation of first peptide bond (“mobile” tRNAs)


α

Large 50S subunit: Peptidyltransferase ribozyme complex

Structural constraints necessitate that both tRNAs likely shift their position in the 50S subunit to assume a hybrid binding states. There is no E site tRNA anticodon binding domain in 30S subunit.

Small 30S subunit: Proofreading occurs after the charged tRNA is in place and both before and after GTP hydrolysis by EF-Tu.

Stage 3: Elongation Step 3: Ribosome translocation to next codon

EF-Tu + tRNA EF-G + GDP

…ready for next cycle of elongation. Note how functional connection between mRNA template and the decoded polypeptide product is maintained.

Facilitates change in ribosome conformation

Peptidyl transfer and translocation likely involves hybrid ribosome states (an idea championed by Harry Noller)

EF-Tu: GDP

Proofreading (ms time scale)

3-nt stepChemistry can happen here.

A look at the putative transition state of peptidyl transferase

Tetrahedral carbon intermediate resolves to yield a deacylatedtRNA (P) and a peptidyl tRNA extended by one amino acid.

P site A site

α3′

Peptidyl transferase inhibitors with P or A site ribosome binding sites.

Adenosine

Puromycin resembles 3′end of amino-acylated tRNA.

P. Nissen et al. Science 289:920-929, 2000

How puromycin inhibits protein synthesis

Structure of peptidyl-puromycin

Puromycin is made by the mold Streptomyces alboniger and affects both prokaryotic and eukaryotic ribosomes.

Ribosome-specific antibiotic inhibitors

• Cycloheximide– Affects 80S ribosome only – Blocks peptidyl transfer

• Streptomycin– Bacterial 30S subunit specific– Causes codon misreading

• Tetracycline– Bacterial A site drug – Block AA-tRNA binding (can’t

pass thru euk cell membranes)• Chloramphenicol

– Affects bacterial, mitochondrial, chloroplast ribosomes only

– Blocks peptidyl transfer• Erythromycin

– Binds bacterial 50S subunit– Blocks elongation/translocation

Atomic view of peptidyl transferase region of Haloarcula marismortui

No proteins near (∼18 angstroms) of active site. Catalytic activity depends entirely on RNA.

Atoms belonging to 23S rRNA >95% conserved in all three kingdoms are red.


Catalytic potential achieved by making A2486 a stronger base via charge relay

3

Negative electrostatic charge originating from buried A2485 phosphate could be relayed to N3 of A2486 via the proposed mechanism to generate an imino tautomer.

Charge relay mechanism is important in serine protease catalysis.

imino

N3 of A2486 is ~3 Å from phosphoramide oxygen and 4 Åfrom amide N.

2



Raising the pKa of A2486 makes the proximal α amino group of AA-tRNA a better nucleophile

3

3

3

A site

P site

N3 represented as standard tautomer but is thought to function as a general base.

Tetrahedral carbon intermediate stabilized by H-bonding between protonated N3 imine group and oxyanion.

Deacylation: Proton transfer from N3 to the peptidyl-tRNA 3′OH.

Stage 4:Termination of polypeptide synthesis

• Signaled by ribosome encountering a stop codon in the A site

• No corresponding stop tRNA (except for Sec-tRNASec) so release factor complex binds to ribosome instead. – RF-1 (UAG, UAA)– RF-2 (UGA, UAA)– RF-3 (GTPase, needed to release 50S

ribosome subunit)• Peptidyltransferase transfers P-site

peptide chain to a water molecule. • Unstable 70S ribosome dissociates

assisted by IF-1 and IF-3 perhaps.• 30S subunit likely stays attached to

polycistronic messages and “slides” to next Shine-Dalgarno sequence.


Mechanisms ↑translational efficiency

This figure is not drawn to scale….. RNA polymerase (Mr ~3.9 ×105) ribosome (Mr ~2.7 × 106)


Translational efficiency enhanced by polysomes too (elongation is rate-limiting)

One ribosome, one mRNA model does not account for the total rate of protein synthesis per E. coli cell.As many as 50 ribosomes bound per mRNA under certain conditions.

Ribosome recycling

In E. coli, 15,000 ribosomes synthesizing @ 15 AA/sec →750 proteins of 300 AA/sec.

Fig. 27.29

Summary of important differences in translation machinery in eukaryotes• Ribosome (polysomes and attached to ER)

– Additional 5.8S rRNA component in large 60S subunit – mRNA aligned on the small 40S subunit using 5′ cap (no

Shine-Dalgarno sequence or fMet)– Scanning identifies correct start Met– No E site, deacylated tRNAs released directly from P site

• Initiation factors (multiple eIFs)– Many more required (~9-11 vs 3)– Some bind mRNA, others attach to ribosomal subunits

• Elongation factors (eEFs)– No differences, all orthologs of prokaryotic EFs

• Termination (only one release factor)– eRF recognizes all stop codons (UAA, UAG, UGA)

Initiation of translation in eukaryotic cells:connecting the head and tail

GCCRCCAUGG

1: Multiple initiation factors with distinct biochemical roles (linking, tethering, recruiting, and scanning)

2: 5′ and 3′ ends of mRNA tied together and tethered to 40S subunit via eIF/PAB complex. Longer poly (A) tract → more efficient translation

3: Identification of start AUG achieved by “scanning” mechanism involving eIF4B and eIF4F(complex of 4E, 4A, and 4G). Initiator tRNA is Met-tRNAMet.

protein synthesis ii biochemistry 302 - university of...

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