Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region; a site-directed cross-linking study with mRNA analogues.

Abstract

Synthetic mRNA analogues were prepared by T7 transcription, each containing several thio‐uridine residues at selected positions. After binding to the ribosome in the presence of cognate tRNA, the thio‐U residues were activated by UV irradiation and the resulting sites of cross‐linking to 16S RNA analysed. Three distinct cross‐links were consistently observed: (i) from position ‘+6’ of the mRNA (the 3′‐base of the A‐site codon) to base 1052 of 16S RNA; (ii) from position ‘+7’ of the mRNA to base 1395; and (iii) from ‘+11’ to base 532. Individual yields of the cross‐links were strongly dependent on the particular mRNA sequence in each case. The ‘+11/532’ and ‘+6/1052’ cross‐links were always entirely tRNA‐dependent, whereas the ‘+7/1395’ cross‐link was observed at lower intensity in the absence of tRNA. In the presence of a second (A‐site bound) tRNA the +6/1052 cross‐link was markedly reduced. A cross‐link to the 1050 region was again observed when a message carrying a thio‐U at position ‘+9’ was translocated on the ribosome so as to bring the thio‐U to position +6. Taken together, the data are incompatible with some current models both for the three‐dimensional arrangement of 16S RNA and for the orientation of the tRNA‐mRNA complex in the ribosome.

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations—implications for the study of ribosome dynamics

“…A1191 (A909) and A1716 (A1413) are among the ''class III'' sites in the 30S subunit that are protected by either binding of tRNA in an mRNA-independent manner, or by binding of the 50S subunit Noller 1986, 1987). In addition, A628 (A532) becomes protected in E. coli 30S ribosomal subunits upon binding of a P-site tRNA and can be directly cross-linked to mRNA (Rinke-Appel et al 1991;Dontsova et al 1992).…”

Complex of Transfer-messenger RNA and Elongation Factor Tu

“…sU is a well known cross-linking reagent used in studies of RNA⅐RNA and RNA⅐protein interactions (17). This reagent can be easily incorporated into RNA molecules by in vitro T7-transcription, and incorporation of sU residues does not significantly affect the structure of the RNA molecules nor their biological activity (36).…”

“…For the synthesis of sU-modified tmRNA transcripts, UTP was supplemented with 4-thio-UTP at different molar ratios (from 1:1 up to 1:100). The level of 4-thiouridine incorporation was estimated using the T2 fingerprinting technique (17). For example, one or two 4-thiouridine residues per molecule were incorporated at an sU:U ratio of 1:100, and tmRNA transcripts were purified as described for mRNAs (23).…”

“…A cross-linking approach developed for the investigation of the ribosomal neighborhoods of mRNA and 5 S rRNA was then applied (17). This method utilizes the photosensitive 4-thiouridine that is incorporated randomly into the RNA under study.…”

Saturation mutagenesis of a +1 programmed frameshift-inducing mRNA sequence derived from a yeast retrotransposon

“…In bacterial ribosomes, G530 contacts both the second and third base pair of the cognate codon d anticodon complex, facilitating its recognition by the ribosome (for review, see Ogle et al 2003). DNA cross-linking assays showed that only when the A site is empty, G530 lies very close to the mRNA region corresponding to the Ty3 stimulator (Dontsova et al 1992;Rinke-Appel et al 1993). Based on the complementarity and proximity of the mRNA and rRNA, Li et al (2001) predicted that the Ty3 stimulator might pair with this region (Fig.…”