Question

6) Please describe the function of the Shine-Dalgarno sequence in prokaryotic translation initiation. (10 points) 

7) During translation elongation in procaryotes, the interplay between EF-Tu and EF-TS regulate tRNA charging to the A site of the ribosome. Please describe in detail the sequence of events that take place during tRNAEFTU GTR ternary complex formation. Explain how the EF-Tu GDP that is discharged from the ribosome is converted to EF- Tu GTR that is the competent complex for translation elongation? What is the role of GTP hydrolysis (GTP bound to EF-Tu) in translation elongation? (15 points)

Answer 1

Answer:

- Shine -dalgarno sequence plays a role in initiation of translation in prokaryotes.

Eukaryotes does not have this sequence instead they have cap protein, poly A tail and kozak sequence

- It is a ribosomal binding site located around 8 bases upstream of the start codon AUG.

-It is the purine rich sequence which binds to pyrimidine rich sequence of ribosomal rRNA.

-Base pairing between these differentiate between starting codon AUG and the internal AUG sequence

-The degree of base pairing plays a role in determining the rate of initiation at different AUG initiator codons in prokaryotes.

- This sequence allows the 30S subunit to start codon, ensure the correct reading frame for the translated protein, and guide the assembly of the large subunit to the small subunit to form an elongation competent 70S complex.

Answer:

- EF-Tu is the elongation factor, catalyze binding of aminoacyl tRNA to A site of ribosome.

-It is a G protein. So uses GTP.

- EF-Tu complex with GTP, which bind to aa-tRNA and form stable complex of EF-Tu-GTP-aa tRNA.

-The binding of an aa-tRNA to EF-Tu • GTP allows for the ternary complex to be translocated to the A-site of an active ribosome, in which the anticodon of the tRNA binds to the codon of the mRNA.

- After that GTP hydrolysis will occur ddue to change in ribosome configuration.

- This alters the geometry of the GTPase domain of EF-Tu, resulting in the hydrolysis of the GTP associated with the EF-Tu to GDP and Pi.

- Ribosome functions as a GTPase-activating protein (GAP) for EF-Tu.-pon GTP hydrolysis, the conformation of EF-Tu changes drastically and dissociates from the aa-tRNA and ribosome complex.

-The aa-tRNA then fully enters the A-site, where its amino acid is brought near the P-site's polypeptide and the ribosome catalyzes the covalent transfer of the polypeptide onto the amino acid.

-In the cytoplasm, the deactivated EF-Tu • GDP is acted on by the prokaryotic elongation factor EF-Ts, which causes EF-Tu to release its bound GDP.

-Upon dissociation of EF-Ts, EF-Tu is able to complex with a GTP due to the 5– to 10–fold higher concentration of GTP than GDP in the cytoplasm, resulting in reactivated EF-Tu • GTP, which can then associate with another aa-tRNA.

-The role of GTP hydrolysis is, it changes confirmation of EF-Tu, so EF-Tu will be relaeased fronm aminoacyl tRNA and ribosome complex.