FCH 532 Lecture 19

This material will not be on the exam

Chapter 32: Translation

TranslationThe participants

Ribosome - ribonucleoprotein enzyme and structural complex

mRNA - carries the DNA information encoding the protein

tRNAs - decoders of mRNA

amino acids and N-formylmethionine

Translation initiation

Initiation complex formation

(30S ribosome subunit-mRNA-fMet-tRNA)

Elongation

Binding charged tRNA

RNA in large ribosomal subunit is the enzyme in peptide bond formation

Translocation

Termination

Stop codons

Release factors

A comparison of the structures of procaryotic and eucaryotic ribosomes.

The ribosome is an allosteric enzyme.The peptidyl-transferase site is the ACTIVE SITE

The Prokaryotic Cast (in eukaryotes similar but more complex)

Ribosome ~3 x 106 Da, 250 Å (50S + 30S = 70S)The ribosome provides the structure necessary for translation

and catalyzes the reaction

What else is needed?

Factors:

• IF 1, 2,3 Initiation

• EF-Tu, EF-Ts, EF-G Elongation

• RF 1, 2, 3, RRF Release

• GTP hydrolysis

Figure 32-41Ribosomal peptidyl transferase reaction forming a peptide bond.

Pag

e 13

20

Figure 32-43Some translational initiation (Shine-Dalgarno) sequences recognized by E. coli ribosomes.

Pag

e 13

21

Shine-Dalgarno sequences typically start 10-15 nt upstream of the initiation codon.

Are only found in prokaryotes.

Figure 32-45Translational initiation pathway in E. coli.

Pag

e 13

23

• 50S and 30S associated.

• IF3 binds to 30S, causes release of 50S.

• mRNA, IF2-GTP (ternary complex), fMet-tRNA and IF1 bind 30S.

• IF1 and IF2 are released followed by binding of 50S.

• IF2 hydrolyzes GTP and poises fMet tRNA in the P site.

Pag

e 13

27

Defining tRNA Binding Sites in Different functional States

GENERATE RIBOSOMES IN THE FOLLOWING STATES:

A (Aminoacyl): EF-Tu.GTP dependent; mRNA dependent; occupied P Site

P (Peptidyl): Reactive with Puromycin (Pm)E (Exit): Deacylated tRNA

MONITOR BY CHEMICAL FOOTPRINTING: 30S A site protections:50S P site protections

(also X-linkers, EDTA-FeII) Looking at footprint pre and post peptide bond, translocation

The data didn't fit into a simple 2 site modelHYBRID STATES HAD TO BE INVOKED

tRNA movement occurs independently on 2 subunits via 6 hybrid states.

1. A/T --> 2. A/A --> 3. A/P --> 4. P/P --> 5. P/E --> 6. E

In this model the tRNA would "ratchet" its way through the ribosome undergoing 50° rotations along its longitudinal axis from A to P.

This model has received support from EM and X-ray studies.

cryo-EM

Pag

e 13

33

EF-Ts

EF-Tu-EF-Ts

GDP

GTP

EF-Ts

EF-Tu-GTP

Aminoacyl-tRNA

Pag

e 13

35

RF-1 = UAARF-2 = UAA and UGA

Cannot bind if EF-G is present.

RF-3-GTP binds to RF1 after the release of the polypeptide.

Hydrolysis of GTP on RF-3 facilitates the release of RF-1 (or RF-2).

EF-G-GTP and ribosomal recycling factor (RRF)-bind to A site. Release of GDP-RF-3

EF-G hydrolyzes GTP -RRF moves to the P site to displace the tRNA.

RRF and EF-G-GDP are released yielding inactive 70S

Translation• Shine-Dalgarno sequence• Initiation• Elongation• Release

Pag

e 13

22