ABSTRACT
Equilibrium binding affinity of yeast tRNA(Phe) for Escherichia coli poly(U)-programmed 70S ribosomal P-site was compared with corresponding affinities of several tRNA(Phe) 3'- and 5'-end-truncated derivatives, all containing the anticodon arm. Our findings strongly suggest that besides three 3'-terminal-CCA nucleotides (C74, C75 and A76), only the tRNA(Phe) anticodon arm (N28-N42) contains ribosomal P-site contact centers and that there are no such centers in the intermediate regions N1-N27 and N43-N73.
Subject(s)
Escherichia coli/metabolism , Poly U/metabolism , RNA, Ribosomal/metabolism , RNA, Transfer, Phe/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Anticodon , Base Sequence , Binding Sites , Escherichia coli/genetics , Kinetics , Molecular Sequence Data , Oligoribonucleotides/chemistry , Saccharomyces cerevisiae/geneticsABSTRACT
Conditions for the reversible binding of isolated anticodon arm of yeast tRNA(Phe) (15-nucleotide, corresponding to nucleotides N28-N42) to the P site of Escherichia coli 30S and 70S ribosomes have been determined. The affinity constant for the anticodon arm binding to 70S ribosomes is shown to be only 30-fold weaker than that for the binding of total tRNA(Phe). The affinities of yeast tRNA(Phe) and the anticodon arm for 30S subunits and of the anticodon arm for the total 70S ribosomes are shown to be equal. These data imply that the anticodon arm moiety of tRNA(Phe) mainly contributes to the tRNA-70S ribosome interaction, i.e., it contributes for the most part to the total free energy of the deacylated tRNA(Phe) interaction with the P site of the 70S ribosome. By taking into account additional contacts in the 3'-CCA end, other contacts in the region besides the anticodon arm and 3'-CCA end are presumably absent. Within the anticodon arm removal of the 3'-end nucleotide corresponding to guanosine-42 in tRNA(Phe) decreases the association constant of the anticodon arm-ribosome interaction 15-fold. Replacement of this guanosine with other nucleosides as well as modification of the ribose moiety (oxidation and reduction) does not affect the affinity. The replacement data are very likely to indicate that G42 affects the anticodon arm affinity by forming a direct contact with ribosome.
Subject(s)
Anticodon , Escherichia coli/genetics , Guanosine/metabolism , RNA, Transfer, Phe/metabolism , Ribosomes/metabolism , Base Sequence , Binding Sites , Kinetics , Molecular Sequence Data , RNA, Transfer, Phe/geneticsABSTRACT
Escherichia coli 70-S ribosomes contain a third site for tRNA binding, additional to the A and P sites. This conclusion is based on several findings. Direct measurements showed that in the presence of poly(U), when both A and P sites are occupied by Ac[14C]Phe-tRNAPhe, ribosomes are capable of binding additionally deacylated non-cognate [3H]tRNA. If ribosomes in the preparation are active enough, the total binding of labeled ligands amounted to 2.5 mol/mol ribosomes. In the absence of poly(U), when the A site can not bind, the P site and the 'additional' site can be filled simultaneously with Ac[14C]Phe-tRNAPhe and deacylated [3H]tRNA, or with [3H]tRNA alone; the total binding exceeds in this case 1.5 mol/mol ribosomes. The binding at the 'additional' site is not sensitive to the template. [3H]tRNA bound there is able to exchange rapidly for unlabeled tRNA in solution. Deacylated tRNA is preferred to the aminoacylated one. The binding of AcPhe-tRNAPhe was not observed there at all. The 3'-end adenosine is essential for the affinity. The function of the 'additional' site is not known, but its existence has to be considered when tRNA . ribosome complexes are studied.
Subject(s)
Escherichia coli/metabolism , RNA, Bacterial/metabolism , RNA, Transfer/metabolism , Ribosomes/metabolism , Acylation , Binding Sites , Kinetics , Poly U/pharmacology , RNA, Transfer, Amino Acyl/metabolismABSTRACT
The tetranucleotide Tp psi pCpGp acts as a specific inhibitor of the rate of AcPhe-tRNAPhe binding in the ribosomal P site. This effect is observed both in the presence and in the absence of poly(U). In the absence of poly(U) antibiotics tetracycline and puromycin also decrease the rate of AcPhe-tRNAPhe binding. Some inhibition by tetracycline is observed with poly(U). All these inhibitors are known to be ligands of the ribosomal A site, and their influence on the P site binding can be most naturally explained by the suggestion that AcPhe-tRNAPhe enters the ribosome via the A site, forms there an intermediate complex, and spontaneous translocation into the P site follows. In the presence of poly(U) arguments in favour of this hypothesis are much weaker, but the same sequence of events is possible.
Subject(s)
Oligonucleotides/pharmacology , RNA, Transfer, Amino Acyl/metabolism , Ribosomes/metabolism , Anti-Bacterial Agents/pharmacology , Binding Sites , Escherichia coli/metabolism , Models, Biological , Phenylalanine/analogs & derivatives , Phenylalanine/metabolism , Poly U/pharmacology , Puromycin/pharmacology , RNA, Bacterial/metabolism , Ribosomes/drug effects , Tetracycline/pharmacologyABSTRACT
fMet-tRNAfMet binds reversibly to the donor site (P-site) of Escherichia coli ribosomes both in the absence of messenger and in the presence of ApUpGp and some other oligonucleotides or poly(U). Kinetics of interaction conforms the second-order law. The equilibrium constants and the rate constants of binding are estimated at 0 degrees C. Not only the cognate trinucleotide ApUpGp but also some other oligonucleotides and even poly(U) stimulate the binding. The presence of total deacylated tRNA considerably increases the selectivity of association.
