Investigation of the tertiary folding ofEscherichia coli16s RNA byin situintra-RNA crosslinking within 30S ribosomal subunits

Abstract

Intra-RNA cross-links were introduced into E. coli 30S ribosomal subunits by mild ultraviolet irradiation. The subunits were partially digested with cobra venom nuclease, followed in some cases by a second partial digestion with ribonuclease H in the presence of the hexanucleotide d-(CTTCCC). The cross-linked RNA complexes were separated by two-dimensional gel electrophoresis and the sites of cross-linking analysed by our published procedures. Tertiary structural cross-links in the 16S RNA were identified between positions 31 and 48, between oligonucleotides 1090-1094 and 1161-1164, and between oligonucleotides 1125-1127 and 1280-1281. The first of these imposes a rigid constraint on the relative orientations of helices 3 and 4 of the 16S secondary structure. A further tertiary cross-link (which could not be precisely localised) was found between regions 1-72 and 1020-1095, and secondary structural cross-links were identified between positions 497 and 545-548, and positions 1238-1240 and 1298.

Protein binding sites onEscherichia coli16S RNA; RNA regions that are protected by proteins S7, S14 and S19 in the presence or absence of protein S9

“…refs. [10][11][12][13]. Cross-linking is a purely topographical approach, which serves to pinpoint sites of contact or neighbourhood within the RNA or between the individual proteins and the RNA, whereas the protection studies indicate RNA regions where an extensive physical interaction with the proteins occurs.…”

Dinoflagellate 17S rRNA sequence inferred from the gene sequence: Evolutionary implications

“…These helices are surrounded by helices 2 and 3, which may be involved in intramolecular switching in the rRNA with helices 28-30 (43). The secondary structure of this region is not clearly defined in eukaryotes, but it is probably organized in a different way than in prokaryotes (usually helix 17 is not stable) (22).…”

RNA PROTEIN CROSSLINKS INTRODUCED INTO E. coli RIBOSOMES BY USE OF THE INTRINSIC PROBE 4‐THIOURIDINE

“…Clearly, the most important parameters determining the eventual formation of a link from a photoactivated s4U and a proteic group are the proximity and orientation factors between the putative reactive groups or atoms as well as the nature of the immediate environment. For example, although the 254-nm photochemistry of nucleic acid residues in solution or in model polynucleotides tell us that by far the pyrimidines are the most reactive, there are numerous recent examples where in the tertiary folding of 16s RNA in the 30s particle Pur-Pur adducts appear to form efficiently (Atmadja and Brimacombe, 1985). Another parameter that should be considered is the excited state life-time of the base, since adequate positioning may only OCCUI transiently.…”

“…In addition to the photoreaction upon the 5-6 double bond already found in photoexcited uridine and cytosine, another photoreactive site is found, namely the C4 sulfur bond. Classically this C-S bond is involved in thietane formation between excited s4U and pyrimidines (Bergstrom and Leonard, 1972) leading to the 8-13 link in tRNA (Favre et al, 1972). Photoreactions relevant to the photoaddition of amino acids have not been extensively studied.…”