DNA-dependent single-step addition reactions catalyzed by Escherichia coli RNA polymerase.

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 reactions A-U + ATP leads to A-U-A and U-A + UTP leads to U-A-U occur in the presence of poly[d(A-T)], while the reactions G-C + GTP leads to G-C-G and C-G + CTP leads to C-G-C take place in the presence of poly[d(I-C)]. These reactions proceed with a turnover of enzyme. The products U-A-U and C-G-C are formed rapidly, while A-U-A and G-C-G are formed much more slowly. Another poly[d(A-T)]-dependent reaction, which occurs with a turnover of enzyme, is U-A-U + ATP leads to U-A-U-A. All of these reactions are only partially inhibited by rifampicin. ATP can be replaced by 3'-deoxyadenosine 5'-triphosphate in the reactions A-U + ATP leads to A-U-A and U-A-U + ATP leads to U-A-U-A, though the rate of formation of the products becomes somewhat slower. The reactions involving 3'-deoxyadenosine 5'-triphosphate are almost completely inhibited by rifampicin, indicating that the 3'-hydroxyl group is necessary for these reactions to occur in the presence of rifampicin.

Antifolate-resistant Chinese hamster cells. Molecular basis for the biochemical and structural heterogeneity among dihydrofolate reductases produced by drug-sensitive and drug-resistant cell lines.

Nucleotide sequence analysis of a cDNA clone shown to direct the synthesis in Escherichia coli of a pI 6.5 form of dihydrofolate reductase (DHFR) with an apparent molecular weight of 21,000 has clarified the allelic nature of the DHFR genes present in the Chinese hamster lung cell line DC-3F. By comparison with other cDNAs encoding different forms of DHFR produced by these cells or by antifolate-resistant sublines derived from them (Melera, P.W., Davide, J.P., Hession, C.A., and Scotto, K.W (1984) Mol. Cell. Biol. 4, 38-48) and with the use of transcription vectors to generate homogeneous populations of specific DHFR mRNAs for subsequent translation in vitro, we demonstrate that, with respect to the proteins they encode, these alleles differ only at amino acid position 95; a conversion of Asp----Asn at this position is solely responsible for the electrophoretic mobility and pI differences between the Mr 21,000 pI 6.5 and Mr 20,000 pI 6.7 forms of the enzyme. We also show that the conversion of Leu to Phe at position 22 of the Mr 21,000 pI 6.5 enzyme results in a mutant form whose catalytic activity is equal to or greater than normal, but whose IC50 for methotrexate is 85 microM. Additionally, the in vitro translation experiments show that the minor pI forms of DHFR known to exist in Chinese hamster lung cells are generated by a translational or post-translational modification step. Preliminary evidence suggests that this modification may result from an acetylation of the N terminus of the protein.

Covalent attachment of a peptidyl-transfer RNA analog to the 50S subunit of Escherichia coli ribosomes.

The peptidyl-tRNA analog, N-bromo-acetyl-Phenylalanyl-tRNA(Phe) 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 peptidyl-tRNA 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.

Identification of 50S proteins at the peptidyl-tRNA binding site of Escherichia coli ribosomes.

Bromoacetyl-phenylalanyl-tRNA(phe) 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-tRNA(phe) can participate in peptidebond formation with phenylalanyl-tRNA(phe) 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 bromoacetylphenylalanyl-tRNA(phe) can function as a normal peptidyl-tRNA and that the 50S proteins, L2 and L27, are located in the peptidyl-tRNA binding site. The side reactions of bromoacetyl-phenylalanyl-tRNA(phe) 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.