The three-dimensional folding of ribosomal RNA; localization of a series of intra-RNA cross-links in 23S RNA induced by treatment ofEscherichia coli50S ribosomal subunits withbis-(2-chloroethyl)-methylamine

Abstract

Intact 50S ribosomal subunits from E.coli were crosslinked with the symmetrical bifunctional reagent bis-(2-chloroethyl)-methylamine. After deproteinization, selected regions of the 23S RNA were excised by treatment with ribonuclease H in the presence of appropriate complementary decadeoxynucleotides, and screened for the presence of intra-RNA cross-links by two-dimensional gel electrophoresis. Individual isolated cross-linked RNA fragments were analysed by our established procedures. Sixteen intra-RNA crosslinks were identified, three of which corresponded to those previously published. The thirteen 'new' crosslinks were localized in the 23S RNA at positions 774

Functional interactions within 23S rRNA involving the peptidyltransferase center

“…Nucleotide 2032 has been cross-linked by nitrogen mustard to 2055 (10). The proximity of these two positions suggests that the 2032A mutation could bring about its effect by altering the tertiary alignment of 2058A and/or the neighboring bases.…”

“…UV and nitrogen mustard cross-links have been created between nucleotides 571 and 2031, 746 and 2613, and 979 to 984 and 2029 (10) (Fig. 1), orienting positions 2032 and 2058 in the midst of domain II.…”

“…The altered tolerances towards chloramphenicol, clindamycin (7-chloro-7-deoxylincomycin), and erythromycin caused by these mutations have been evaluated and compared with resistances conferred by domain II (20). Arrows indicate the relevant cross-linked (X-link) bases (10) and the positions photoaffinity labeled by benzophenone-derivatized Phe tRNA (PB-Phe tRNA) (1) andp-azidopuromycin (6). Sites within this region where interaction of the tRNA 3'-end causes altered reactivities to chemical reagents (tRNA 3-end footprint) are shown (26).…”

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

“…A number of different biochemical methods have been applied to the study of the 3D folding of the large ribosomal RNA molecules, and the data obtained have been incorporated into crude models or "cartoons" representing the 3D arrangement of the double-helical elements of the Escherichia coli 16S (e+g+, Brimacombe et al+, 1988;Stern et al+, 1988;Malhotra & Harvey, 1994) or 23S (Nagano et al+, 1988;Mitchell et al+, 1990) rRNA+ More recently, rapid advances in the cryo-electron microscopy (EM) of ribosomal particles combined with computerized image processing techniques have led to the derivation of 3D structures of the E. coli 70S ribosome at a resolution of ca+ 20 Å (e+g+, Stark et al+, 1995Stark et al+, , 1997aAgrawal et al+, 1996)+ At this resolution, ligands such as tRNA or elongation factor EF-Tu can be directly visualized (Stark et al+, 1997a(Stark et al+, , 1997b, and many fine structural elements are discernible that have dimensions suggestive of RNA double helices+ By combining the rRNA modeling studies with these new EM structures, it has been possible in the case of the 16S rRNA to fit the rRNA model to the EM contour Mueller et al+, 1997), thus giving the previous "cartoon" models for the first time a detailed physical basis+ Although the resolution of the EM structures is continuously improving, there are still a number of areas of uncertainty or ambiguity in the 16S rRNA structure, where additional biochemical information relating to the 3D arrangement of the rRNA helices would be very helpful+ In the case of the 23S molecule, where the 3D models are considerably more primitive (Nagano et al+, 1988;Mitchell et al+, 1990) and have yet to be fitted to the EM contour, the need for this type of information is even greater+ Paradoxically, whereas the older types of approach to the problem, such as in situ intra-RNA crosslinking using short bifunctional reagents (e+g+, Döring et al+, 1991) or direct UV irradiation (Stiege et al+, 1986), have yielded relatively high-resolution data, some of the methods that have been applied more recently offer only a significantly lower level of resolution+ For example, Fe(II)EDTA tethered to the ends of tRNA anticodon loop analogues has been used to generate hydroxyl radical cleavages in the rRNA (Joseph et al+, 1997); here the length of the tether combined with the diffusion distance of the hydroxyl radical gives cleavages lying within a large (40-50 Å diameter) sphere encompassing the site of the probe (Joseph & Noller, 1996)+ In another approach, complementary oligodeoxynucleotides carrying a photolabel at one end have been hybridized to single-stranded hairpin loop-ends in the rRNA, and the resulting sites of attachment to the photolabel have been analyzed (e+g+, …”

“…We recently described a new method for studying the 3D topography of the rRNA, whereby a photoreactive 4-thiouridine ("sU") residue in the form of its 39, 59-biphosphate ("psUp") is inserted with the help of RNA ligase at a specific cleavage site in the rRNA (Baranov et al+, 1997); the cleavages (in 16S rRNA) were induced in isolated rRNA by RNase H in the presence of complementary chimeric oligonucleotides+ A similar approach was developed simultaneously by Yu and Steitz (1997), using mRNA analogues+ Ideally, one would want to religate the cleaved rRNA so as to obtain a continuous RNA chain [as was done by Yu & Steitz (1997) in their experiments], but religation of the large rRNA molecules (as opposed to mRNAs) in reasonable yield has proved to be extremely difficult (Bogdanova et al+, 1995)+ Fortunately, however, it is possible in many cases to reconstitute cleaved 16S rRNA into 30S ribosomal subunits (Afonina et al+, 1991a(Afonina et al+, , 1991b, and these subunits are able to re-associate with 50S subunits to form 70S ribosomes in a normal manner (Baranov et al+, 1997)+ After photoactivation of the psUp residue (which at the same time is radioactively labeled with 32 P), the sites of crosslinking from the sU can be determined within the rRNA+ Although the method does not offer quite such a high level of resolution as the older intra-RNA crosslinking methods already mentioned (Stiege et al+, 1986;Döring et al+, 1991), it has, on the other hand, the distinct advantage that the site for the insertion of the psUp residue can be chosen by the investigator; thus, the environment of any desired area of the rRNA can be studied, provided, of course, that the cleavage site does not inhibit the subsequent reconstitution of the rRNA into ribosomal subunits+…”