3. To study bacterial ribosomes, researchers make use of antibiotics that inhibi

Question

3. To study bacterial ribosomes, researchers make use of antibiotics that inhibit ribosome function at different steps of translation. What would be present (uncharged-tRNA, amino acyl-tRNA, peptidyl-tRNA, etc..) in the A and P sites of an elongating prokaryotic ribosome when the following antibiotics are present. a. Tetracycline: binds in the A site a blocks entry of amino-acyl tRNA to the A site of ribosome. b. Kanamyocin: prevents translocation. c. Kirromycin: prevents release of EF-Tu/GDP from aa-tRNA d. Puromycin: can bind the ribosome peptidyl transfer active site and accept the growing peptide chain. e. Ricin: cleaves the rRNA, which inactivates its GAP function.

Explanation / Answer

Answer:
a. Tetracycline: This binds to the A-site of the ribosome and blocks the entry of aminoacyl t-RNA to the A-site. Hence, the peptide synthesis gets halted and we are left with the unfinished peptidyl t-RNA and a charged t-RNA.

b. Kanamyocin: During protein synthesis, the incoming charged amino-acyl t-RNA binds to the A-site and then the t-RNA translocates to the P-site progressively and then from P-site to the E-site, during which the peptide chain gets shifted from the t-RNA which is in P-site to the incoming new amino-acid which is attached to the t-RNA at the A-site. So, the process of translocation happens from A-site to P-site and from P-site to E-site. This antibiotic prevents translocation, thereby leaving us with an unfinished peptidyl t-RNA and an A-site bound aminoacyl t-RNA.

c. Kirromycin: This prevents release of the EF-Tu GDP from aa-tRNA. The aminoacyl t-RNA EF-Tu GTP brings the aminoacyl t-RNA to the A-site of the ribosome. After its deposition on the A-site, GTP is cleaved to from GDP and phosphate. This EF-Tu/GDP dissociate from the ribosome. The aminoacyl t-RNA has a lower affinity to EF-Tu/GDP and a higher affinity to the A-site, so that it stays on the ribosome and the EF-Tu/GDP would dissociate from the t-RNA and from the ribosome. Kirromycin prevents this disassociation of aminoacyl t-RNA bound to EF-Tu/GDP, and hence we are left with the unfinished peptidyl t-RNA and A-site bound to EF-Tu/GDP with the aminoacyl t-RNA.

d. Puromycin: Being a structural analogue of 3'end of amino-acyl t-RNA, binds to the A-site of the ribosome peptidyl transferase center. When puromycin binds to A-site, peptidyl transferase enzyme links the peptide residue of the peptidyl t-RNA which is on the P-site of the ribosome to puromycin via covalent bonds. And no further peptidyl transfer can take place.
Hence we are left with peptidyl-puromycin complex, which snaps off from the ribosome.

e. Ricin: cleaves the rRNA thereby inactivating the GTPase activating protein function. Ricin is responsible for cleavage of a glycosidic bond within the large rRNA of the 60S subunit of ribosomes. The GTP to GDP formation is inhibited, thereby leaving us with aminoacyl t-RNA EF-Tu/GTP at the A-site and the disassociation of aminoacyl t-RNA from EF-Tu/GTP doesn't take place. At the end, we have unfinished peptidyl t-RNA and A-site bound to EF-Tu/GTP with the aminoacyl t-RNA.

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