Identification of 50S Proteins at the Peptidyl-tRNA Binding Site of Escherichia coli Ribosomes

Abstract

Bromoacetyl-phenylalanyl-tRNAPhe bound to 70S E. coli ribosomes reacts covalently with proteins of the 50S subunit. The major reactions are with proteins L2 and L27. In the presence of poly(U), 70S-bound bromoacetyl-phenylalanyl-tRNAPhe can participate in peptidebond formation with plhenylalanyl-tRNAPhe or puromycin.Most of the products of these reactions are also found covalently attached to L2 and L27. Chloramphenicol and sparsomycin markedly inhibit the peptide-bond formation. These results strongly suggest that bromoacetylphenylali.nyl-tRNAPhe can function as a normal peptidyltRNA and that the 50S proteins, L2 and L27, are located in the peptidyl-tRNA binding site. The side reactions of bromoacetyl-phenylalanyl-tRNAPhe are with one or more 50S proteins from the set L14-17, L6 and/or L11, and L26.These occur to a much less extent than the reactions with L2 and L27. Any functional significance of the side reactions is unknown.Previously we described the preparation and some of the properties of the peptidyl-tRNA analogue, N-bromoacetylphenylalanyl-tRNAPhe (BrAcPhe-tRNA). This compound was designed as a potential affinity label for the P site of Escherichia coli ribosomes (1). When bound to 70S ribosomes with poly(U) as messenger, this analogue reacted covalently with the 50S subunit. Initial studies suggested that there were two sites to which the reagent became covalently attached. BrAcPhe-tRNA was found associated with a single band in a one-dimensional polyacrylamide gel separation of the 50S proteins. This band contains proteins 3VI, 2IX, and 2XI [numbering according to Traut et al. (2)]. BrAcPhe-tRNA was also found associated with 23S rRNA. This finding implied either covalent attachment to the RNA or to a protein tightly bound to the RNA.We have now been able to define more clearly the sites of covalent attachment of BrAcPhe. Two-dimensional gel electrophoresis has been used to obtain fairly unambiguous identification of the covalently modified 50S proteins. Also, improved procedures lead to a 30-fold increase in

The proteins of donor tRNA‐binding site of Escherichia coli ribosomes

“…Protein S19 takes part in IF-dependent fMettRNA Met binding to ribosomes [ 16]. Participation of the 50 S proteins L11, L13, L14 and L27 in P-site formation was shown using different tRNA derivatives containing reactive groups in the aminoacyl residue [17,18]. To date, several models are known of 30 S ribosomal subunit from E. coli obtained by different methods [19][20][21][22] for describing positions of proteins on the surface of the 30 S subunit.…”

The primary structure of protein L27 from the peptidyl‐tRNA binding site of Escherichia coli ribosomes

“…Studies with different affinity analogues of peptidyl-tRNA [1][2][3] led to the identification of protein L27 as one of the major products labelled with the analogue. Thus, protein L27 can be inferred to be very close to the peptidyl-tRNA binding site (P-site).…”

Part of the 23S RNA located in the 11S RNA fragment is a constituent of the ribosomal peptidyltransferase centre

“…There is, however, circumstantial evidence to support the concept that a region located within the 11s RNA fragment participates in the peptidyltransferase centre. Protein L27, a protein labelled by a variety of affinity labels which are specific for the peptidyltransferase centre [3,7,9], has a binding site located within the region of the 11s RNA fragment as identified by direct binding studies [la] Fig.3. Limited hydrolysis of photoaffinity labelled 50s subunits.…”