The addition of a single nucleotide to a short oligonucleotide, catalyzed by RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) in the presence of synthetic DNA templates, has been studied. The

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

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The peptidyl-tRNA analog, N-bromoacetyl-Phenylalanyl-tRNAPhe has been prepared. Its binding to the 70S ribosome of E. coli is totally dependent upon polyuridylic acid. The analog becomes covalently attached to the 50S particle. It is associated with only one protein fraction after polyacrylamide-gel separation of total 50S proteins. The analog also reacts with 23S ribosomal RNA or a protein that remains tightly bound to this RNA after treatment with LiCl-urea and sodium dodecyl sulfate. The analog can function as a peptidyltRNA for at least one peptide transfer, but it then inhibits further chain elongation. This result strongly suggests that this analog becomes covalently bound at the P-site of the ribosome. 70SRibosomes of Escherichia coli contain more than 50 different components. The exact structural and functional role of any of these components in protein synthesis is not known with clarity. While a great deal of progress has been made in characterizing the location and function of 30S proteins (1), much less is known about the proteins of the 50S particle. At least one central step of protein synthesis, the formation of peptide bonds, is localized exclusively on the 50S particle (2, 3). The critical component(s) for this function, peptidyl transfer, have not been unambiguously identified. In this work, we report an effort to identify a 50S ribosomal protein that is located adjacent to the peptide group of ribosome-bound peptidyl-tRNA. Such a protein would be a likely candidate to be peptidyl transferase.The approach used in this work is an extension of the idea of affinity labeling (4). In studies of individual enzymes, substrate analogs bearing a chemically reactive group have been used to probe the location of residues at or near an active site. In the present work, the protein-reactive group is attached to a tRNA rather than to a small molecule. Initially, we are interested in determining to which of the ribosomal components this reagent can couple. The identification of the particular groups within a protein or RNA molecule that have been labeled is reserved for future studies. It should be evident that affinity labeling in this manner can provide a critical bridge between the relatively imprecise kind of structural information available about a large particle like the ribosome and the high-resolution structural data accessible for single proteins. On the other hand, for a structure as complex as the ribosome, it is not clear how specifically defined a given binding site will be. The product of this reaction was dissolved in dimethyl- METHODS AND RESULTS Ribosomes

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