Subject(s)
Polylysine/biosynthesis , RNA, Transfer, Amino Acyl/metabolism , Ribosomes/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Kinetics , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Transfer, Amino Acyl/genetics , Templates, GeneticABSTRACT
We have demonstrated that in certain conditions 50S subunits can transfer peptide moiety from peptidyl-tRNA to puromycin in the absence of alcohol. Monovalent cations NH4+ and K+ support this reaction, while Na+ and Li+ are ineffective. Optimal concentration for NH4+ is 1.8 M. Mg2+ ion concentrations above 12 mM are needed as well as an elevated temperature (30 degrees C). Using the alcohol-free puromycin reaction of 50S subunits we show that alcohol activates the peptidyl transferase center, but does not participate in the puromycin reaction. Neither does it change the protein composition of subunits.
Subject(s)
Acyltransferases/metabolism , Alcohols/pharmacology , Escherichia coli/metabolism , Peptidyl Transferases/metabolism , Puromycin/metabolism , Ribosomes/metabolism , Binding Sites , Enzyme Activation/drug effects , Escherichia coli/enzymology , Methanol/pharmacology , Puromycin/analogs & derivatives , Ribosomes/enzymologyABSTRACT
We show that Escherichia coli 50S ribosomal subunits depleted of protein L16 can nevertheless catalyze the transfer of the peptide moiety from fMet-tRNA to puromycin, being, however, unable to use a fragment CACCA-Phe as an acceptor substrate. On the other hand, we found that protein L16 as well as its large fragment (amino acids 10-136) both interact with tRNA in solution (Kd approximately 10(-7) M). Moreover, L16 interacts with CACCA-Phe in solution as well as protects 3' end of tRNA from the enzymatic degradation. We suggest that L16, although not being the peptidyl transferase as such, is involved in the binding of the 3' end cytidines of tRNA into the ribosomal A site.
