Mapping labeled sites in Escherichia coli ribosomal RNA: distribution of methyl groups and identification of a photoaffinity-labeled RNA region putatively at the peptidyltransferase center

Abstract

We have developed a method for the rapid localization of sites of ribosomal RNA labeling to limited regions (approximately 200 bases). The method is based on the formation and polyacrylamide gel electrophoretic separation of hybrids between restriction fragments of rrnB DNA and isotopically labeled rRNA and the subsequent determination of radioactivity across the gel. Using [3H]adenine-labeled rRNA as a control sample, we optimized experimental conditions with respect to a number of variables, including rRNA:DNA stoichiometric ratio, temperature of the annealing step, and levels of nucleases. An important result is that different rRNA X DNA hybrid fragments are obtained in different yields. The method was then applied to analyses of C3H3-labeled rRNA, giving results in good accord with known and proposed sites of rRNA methylation, and of rRNA that has been photoaffinity-labeled with 5-azido-2-nitrobenzoyl-[3H]Phe-tRNAPhe, a probe directed toward the peptidyltransferase center. The latter study showed a single major site of RNA labeling, falling within bases 2445-2668 of 23S rRNA. The extent of labeling was shown to be dependent on light-induced formation of a reactive intermediate and to be decreased in the absence of poly(uridylic acid) or in the presence of puromycin. The location of this major site of labeling is consistent with recent results obtained with an analogous tRNA photoaffinity label [Barta, A., Steiner, G., Brosius, J., Noller, H. F., & Kuechler, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3607-3611] and with related genetic and biochemical studies of antibiotic interaction with ribosomes suggesting that the peptidyltransferase center falls within region V (bases 2043-2625) of 23S rRNA.

Reconstitution of Escherichia coli ribosomes containing puromycin-modified S14: functional effects of the photoaffinity labeling of a protein essential for tRNA binding

“…The 70s ribosomes were prepared from E. coli Q13 as described by Jaynes et al (1978) as method T. Ribosomal subunits were isolated as previously described (Goldman et al, 1983). The 16s rRNA was prepared from 30s subunits essentially as described by Hall et al (1985) except that bentonite was omitted and rRNA was precipitated 3 times by incubation with ethanol for 1 h to remove all traces of phenol.…”

Multiple crosslinks of proteins S7, S9, S13 to domains 3 and 4 of 16S RNA in the 30S particle

“…(Table 2 & 3). With EDC the contacts corresponding to sections 5, 6, 7 were also detected (17). S7 was already found crosslinked at three very different sites: the first being at position 1378 (18) situated at the junction of sections 5 and 6; the second site is at position 1240 (19) in section 4, the third at position 1265 (4) In section 4 These sites probably correspond to those detected here with sections 4, 5 and 6 which represent 9.6% (section 4), 17% (section 5) and 44% (section 6) of the total S7 linked in domains 3 and 4…”

“…The DNA probe fullfills two functions; it first selects a small section in a large RNA molecule and second protects it from RNAses used to eliminate the non selected sections. The use of cloned DNA fragments has recently been described by several groups for selection of fragments of E co/i large ribosomal RNAs, for secondary and tertiary structure analysis of the 58/515 binding domain (5) as well as for localization of peptidyl transferase center by modified aminoacyl tRNA (6)(7) or chemical probing of RNA conformation (8). All these probes were double stranded DNA derived from the plasmid pKK 3535 (9).…”

Cross-linking of streptomycin to the 16S ribosomal RNA of Escherichia coli

“…sites of the 16S RNA which are cross-linked to the antibiotic, we have formed hybrids between restriction fragments of the 16S RNA gene and 16S RNA cross-linked to [3H] dihydrostreptomycin, using a procedure developed by Hall et al (1985). Single-stranded RNA and DNA were digested with a single-strand-specific nuclease, the hybrids were fractionated by gel electrophoresis, and the radioactivity in the hybrid bands was counted.…”