Partial release of AcPhe-Phe-tRNA from ribosomes during poly(U)-dependent poly(Phe) synthesis and the effects of chloramphenicol.
Poly(U)-programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl-tRNA analogue AcPhe-tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe-tRNA participate in poly(U)-directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH4+. Here it is demonstrated that the apparently 'inactive' ribosomes (greater than or equal to 35%) are able to participate in peptide-bond formation, but lose their nascent peptidyl-tRNA at the stage of Ac(Phe)n-tRNA, with n greater than or equal to 2. The relative loss of early peptidyl-tRNAs is largely independent of the degree of initial saturation with AcPhe-tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe)n-tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH4+, 2 mM spermidine, 0.05 mM spermine). Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe-tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2-tRNA nor peptide bond formation between AcPhe-tRNA and Phe-tRNA. The drug facilitates the release of Ac(Phe)2-4-tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).
Rheinberger
H J
HJ
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin-Dahlem, Federal Republic of Germany.
Nierhaus
K H
KH
D001665:Binding Sites; D002701:Chloramphenicol / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D010452:Peptide Biosynthesis; D010455:Peptides; D011072:Poly U / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects; D011691:Puromycin / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*
D010455:Peptides; D012346:RNA, Transfer, Amino Acyl; D012360:RNA, Transfer, Phe; C066516:tRNA, N-acetylphenylalanyl-phenylalanine-; D011072:Poly U; C012001:polyphenylalanine; D011691:Puromycin; D002701:Chloramphenicol