Determinants of peptidyl-tRNA drop-off from E. coli Ribosomes : Effects of antibiotics, mutations and translation factors

Sammanfattning: During protein synthesis, some peptidyl-tRNA molecules dissociate from the ribosomes and become hydrolysed by peptidyl-tRNA hydrolase (Pth). Two different in vitro systems, poly(U) and natural mRNA programmed ribosomes, have been used in order to determine dissociation (drop-off) rates of deacylated- and peptidyl-tRNAs from different sites on ribosomes.A direct measurement of the rates of dissociation of dipeptidyl-tRNAs from the A- and P-site of poly(U) programmed wild-type (wt), hyper-accurate (SmD and SmP, altered in ribosomal protein S12) and error-prone (ram, altered in ribosomal protein S4 or S5) ribosomes has been performed. Dipeptidyl-tRNA dissociates faster from the A-site of wt than from ram ribosomes and it dissociates faster from SmD and SmP than from wt ribosomes. The dissociation of dipeptidyl-tRNA from the P-site has an opposite behaviour compared to the A-site. Dipeptidyl-tRNA dissociates faster from the P-site of ram than from wt ribosomes and faster from wt than SmD and SmP ribosomes. Aminoglycoside antibiotics [streptomycin (Sm)and neomycin (Nm)] reduce the dissociation rate of dipeptidyl-tRNA from the A-site and increase it from the P-site for all these ribosomal variants. Remarkably, when the binding of dipeptidyl-tRNA to the A-site is weak, the affinity for the P-site is strong, and vice versa. In the absence of Sm non-cognate dipeptidyl-tRNA dissociates 150 times faster from the A-site of ram ribosomes than cognate. In the presence of Sm the rate difference is only eight fold. This suggests that Sm may distort the 16S rRNA conformation and, consequently, the codon-anticodon structure.Using a new in vitro system with heteropolymeric mRNAs has revealed that i) Initiation factors IF1 and IF2 synergistically remove short peptidyl-tRNAs from the P-site of ribosomes in a way that depends both on the length and sequence of the nascent peptide. ii) Ribosome recycling factor (RFF), elongation factor G (EF-G) and release factor 3 (RF3) stimulate dissociation of short peptidyl-tRNAs from the P-site in almost the same manner as IF1/IF2. iii) RRF, EF-G, GTP and initiation factor 3 (IF3), after termination, split the 70S ribosomes and remove deacylated tRNA from the P-site of ribosomes in a mechanism that is strictly dependent on GTP hydrolysis. In vivo experiments, showing that overexpression of RRF or simultaneous overexpression of IF1/IF2 impairs growth rate in thermosensitive pth mutant strains, are consistent with the in vitro results.