Identification of 23S rRNA nucleotides neighboring the P-loop in the Escherichia coli 50S subunit

Abstract

We report the synthesis of a radioactive, photolabile 2'-O-methyloligoRNA probe, 2258-53/52(SAz)-48, PHONT1, and its exploitation in identifying 23S rRNA nucleotides neighboring the so-called 'P-loop'. The probe is complementary to nt 2248-2258 in Escherichia coli 50S subunits. PHONT1 contains a p-azidophenacyl group attached to a phosphorothioate bridge between the nucleotides complementary to the positions 2252-2253, such that the photogenerated nitrene is maximally 17-19 A from 23S RNA nucleotides G2252 and G2253. PHONT1 binds to the 50S subunit, and photoincorporates within or immediately adjacent to its target site, as well as into several nucleotides falling between G2357 and A2430. The significance of these results for the structure of the peptidyl transferase center is considered. The PHONT approach is generally applicable to studies of complex RNA-containing molecules.

Identification of 50S Components Neighboring 23S rRNA Nucleotides A2448 and U2604 within the Peptidyl Transferase Center of Escherichia coli Ribosomes

“…The corresponding photoincorporation into TP50, as estimated from total radioactivity coeluting with 50S protein on RP-HPLC analysis, was lower and much less sitespecific. This pattern of higher site-specificity in RNA vs protein labeling is typical of our results with photolabile oligonucleotides (4)(5)(6)(7)(8)18). Little if any photoincorporation into RNA or protein was observed on irradiation of either 32 P-2′-O-methyloligoRNA 2612-04 or 32 P-2′-O-methyloli-goRNA 2458-48 with 50S subunits, demonstrating the aryl azide-dependence of the photoincorporation process.…”

“…Except as specified below, materials were obtained and methods were performed as described elsewhere (4)(5)(6)(7)(8)18).…”

“…Chemical Footprinting. The procedures utilized followed Moazed et al (19) as modified in (8). In experiments measuring the effects of added oligonucleotides on chemical footprinting, the oligonucleotide/50S molar ratio was ten.…”

“…We have been using radioactive photolabile oligonucleotide probes (PHONTs) targeted toward functionally important sites of 23S rRNA to identify proteins and individual nucleotides site-specifically photolabeled from these sites ( − ). Such information is important for the related processes of detailed model building and structure determination.…”

Photolabile anticodon stem–loop analogs of tRNAPhe as probes of ribosomal structure and structural fluctuation at the decoding center

“…Kethoxal modification on ribosomal complexes containing P-site bound tRNA f Met or ASL was performed as described (Moazed and Noller 1986) with slight modifications (Bukhtiyarov et al 1999;Seo and Cooperman 2002). The sites of 16S rRNA modifications were analyzed by primer extension assay (Cooperman et al 2000) using the oligodeoxynucleotide complementary to 16S nucleotides 984-969 as the primer.…”