Dissociation rates of peptidyl-tRNA from the P-site of E.coli ribosomes.
We studied the dissociation rates of peptidyl-tRNA from the P-site of poly(U)-programmed wild-type Escherichia coli ribosomes, hyperaccurate variants altered in S12 (SmD, SmP) and error-prone variants (Ram) altered in S4 or S5. The experiments were carried out in the presence and absence of streptomycin, and the effects of neomycin were tested in the wild-type ribosomes. Binding of peptidyl-tRNA to the P-site of wild-type ribosomes is much stronger than to their A-site. Addition of streptomycin dramatically reduces its affinity for the P-site. The S12 alternations make the P-site binding of peptidyl-tRNA much tighter, and the S4, S5 alterations make it weaker than in the case of the wild-type. We find that when binding of peptidyl-tRNA to the A-site is weak, then the affinity for the P-site is stronger, and vice versa. From these results, we formulate a hypothesis for the actions of streptomycin and neomycin based on deformations of the 16S rRNA tertiary structure. The results are also used to interpret some in vivo experiments on translational processivity.
Karimi
R
R
Department of Molecular Biology, BMC, Box 590, S-751 24, Sweden.
Ehrenberg
M
M
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D013307:Streptomycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012346:RNA, Transfer, Amino Acyl; D012269:Ribosomal Proteins; C034212:ribosomal protein S12; C031798:ribosomal protein S4; C030831:ribosomal protein S5; C019037:tRNA, peptidyl-; D009355:Neomycin; D013307:Streptomycin