The path of mRNA through the Escherichia coli ribosome; site-directed cross-linking of mRNA analogues carrying a photo-reactive label at various points 3′ to the decoding site.

Abstract

mRNA analogues approximately 40 bases long were prepared by T7 transcription from synthetic DNA templates. Each message contained the sequence ACC‐GCG (coding for threonine and alanine, respectively), together with a single thio‐U residue located at a variable position on the 3′‐side of these coding triplets. The thio‐U residue was either substituted with 4‐azidophenacyl bromide to introduce a photo‐reactive group, or was left unsubstituted for direct UV cross‐linking. After binding to Escherichia coli 70S ribosomes in the presence of tRNA‐Thr or tRNA‐Ala, the thio‐U residue or azidophenyl group was photo‐activated and the products of cross‐linking (which was exclusively to the 30S subunit) were analysed. Immunological analysis of the cross‐linked proteins showed that S5 and S3, together with S1, were the targets of cross‐linking at positions close to the decoding site, with the cross‐linking to S3 and S1 persisting at positions further away. Analysis of the 16S RNA showed cross‐links to the region of bases 1390–1400 in all cases, but in one instance (with the reactive nucleotide 11 bases from the decoding site) simultaneous cross‐linking was observed to the latter region and to position 532; these two RNA regions are far apart in current three‐dimensional models of the 30S subunit.

Ribosomal localization of the mRNA in the 30S initiation complex as revealed by UV crosslinking

“…It is noteworthy that the main mRNA crosslinking target on the 16S RNA identified in this paper is compatible with one of the two positions (i.e. nucleotide 532) found covalently linked to the mRNA by RinkeAppel et al [16]; however° other regions of the 16S RNA (i.e. position 1050 [24], 1390-1400 [16,25,26] and the Y-terminus [24,26]) which have been implicated in mRNA-ribosome interaction in other studies were found to be crosslinked to the mRNA either very weakly or not at all (spot 11 of Fig.…”

“…Based on our current knowledge, in this rRNA region, the 530 loop is the most likely candidate for an interaction with the mRNA. In fact, this universally conserved segment of the 16S RNA constitutes a functional pseudoknot [1(3] implicated in and probably stabilized by protein S12 binding [11,i2], involved in A-site decoding [13], streptomycin sensitivity [14o15], mRNA binding [16] and possibly in monitoring translational reading frame [17]. Furthermore, based on a number of considerations summarized by Brimacomb¢ [18], it is now suggested that the methylated G at position 527, which belongs to the 530 loop region, is in a tight cluster with almost all other modified bases of the rRNA.…”

“…Thus, nearly all the elements of the 16S rRNA thought to be involved in or to influence the mRNA binding and decoding (i.e. the 530 loop [16], the Y-end (A1542) [21][22][23], the anti-SD sequence ( 1534-1541) [2] and the nucleotide 1400 region [19,20]) would be located in close proximity to each other in the platform/cleft region of the 30S subunit. It is noteworthy that the main mRNA crosslinking target on the 16S RNA identified in this paper is compatible with one of the two positions (i.e.…”

“…nucleotide 532) found covalently linked to the mRNA by RinkeAppel et al [16]; however° other regions of the 16S RNA (i.e. position 1050 [24], 1390-1400 [16,25,26] and the Y-terminus [24,26]) which have been implicated in mRNA-ribosome interaction in other studies were found to be crosslinked to the mRNA either very weakly or not at all (spot 11 of Fig. 1, for instance, corresponds to nucleotides 1385-1542).…”

New features of 23S ribosomal RNA folding: The long helix 41–42 makes a “U-turn” inside the ribosome

“…The 50S subunits reconstituted from the four fragmented 23S rRNA species carrying 32 P-labeled psUp at the cleavage sites were separated by sucrose gradient centrifugation and subjected to mild UV irradiation at 350 nm (Rinke-Appel et al+, 1991; Dontsova et al+, 1992)+ The rRNA was then isolated from the irradiated subunits (or from nonirradiated control subunits) by phenol extraction, and the crosslinked regions within the rRNA were localized using the RNase H (Dontsova et al+, 1991;Rinke-Appel et al+, 1991)+ For this purpose, either two or three oligodeoxynucleotides (10-20 nt long) complementary to the 23S rRNA were necessary in the RNase H digestions+ The first oligodeoxynucleotide is complementary to a region upstream of the psUp site, so as to release a short rRNA fragment carrying the 32 P-labeled psUp residue+ This oligodeoxynucleotide is kept constant in the RNase H digestions, whereas the second one (and the third one, if present) is varied so as to excise different sections of the 23S rRNA encompassing the crosslinked target site (or sites)+ In contrast to the digestions with the chimeric oligonucleotides used to generate the initial specific cuts in the rRNA, these RNAse H digestions are performed at 558 with an excess of enzyme (cf+ RinkeAppel et al+, 1991)+ Candidates for the "constant" oligodeoxynucleotide were carefully selected so as to obtain a clean and quantitative digestion in every case+ Thus, in the subsequent gel electrophoresis of the rRNA fragments released by the RNase H, the lanes from the control (nonirradiated) 50S subunits should only show the short fragment carrying the labeled psUp in each case, whereas the lanes from the irradiated subunits should, in addition, show products corresponding to this short fragment crosslinked to the fragments of variable length excised from the target region+ Examples of these gels are illustrated in Figure 3, and will be described below for the individual crosslinks+ When the locations of the crosslink sites had been narrowed down in this way to rRNA regions of less than ca+ 50 nt, the sites of crosslinking within the regions concerned were analyzed by primer extension (Moazed et al+, 1986)+ The substrates for the primer extension reactions were isolated from RNase H digestions on a preparative scale+ For this purpose, the digestions were chosen so as to release the crosslinked material as a relatively large rRNA complex of 100-200 nt (cf+ Fig+ 3A, lane 5, or Fig+ 3B, lane 1), and a control fragment was extracted from the same gel lane in each case+ These control fragments represent the noncrosslinked rRNA from the target region and do not contain the region with the radioactive psUp residue; they could accordingly be located by UV shadowing as bands moving just below the corresponding crosslinked complexes in the gels+ Examples of the primer extension gels obtained from the crosslinked and control fragments in the various cases are shown in Figure 4, and are described below for the individual crosslinks+ As a result of the artifacts that are often observed in primer extension analyses of crosslink sites (see Brimacombe, 1995, for discussion), we only consider a stop signal in the reverse transcriptase gels as being indicative of a crosslink site if it is reproducibly observed within the short rRNA region already defined by the RNase H digestions (cf+ Fig+ 3)+…”