The localization of multiple sites on 16S RNA which are cross-linked to proteins S7 and S8 in Escherichia coli 30S ribosomal subunits by treatment with 2-iminothiolane

Abstract

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by reaction with 2-iminothiolane followed by a mild ultraviolet irradiation treatment. After removal of non-reacted protein and partial nuclease digestion of the cross-linked 16S RNA-protein moiety, a number of individual cross-linked complexes could be isolated and the sites of attachment of the proteins to the RNA determined. Protein S8 was cross-linked to the RNA at three different positions, within oligo-nucleotides encompassing positions 629-633, 651-654, and (tentatively) 593-597 in the 16S sequence. Protein S7 was cross-linked within two oligonucleotides encompassing positions 1238-1240, and 1377-1378. In addition, a site at position 723-724 was observed, cross-linked to protein S19, S20 or S21.

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

“…Cross-links D and E both lie within the "head" region, and, particularly cross-link E, provide important restrictions on the possible orientations of helices 30 through 41. Combined with the known sites of cross-linking to protein S7 in helices 41 (22) and 43 (23), the data begin to give a picture of the detailed threedimensional arrangement of the RNA in the head of the 30S subunit (see ref. Once again, as in the Introduction, we emphasize that all of the cross-links described here were induced in situ within the ribosomal subunits, and should be contrasted with the work of other research groups, who are currently attempting to investigate the tertiary structure of the ribosomal RNA by cross-linking of isolated RNA (5,8,9; see also refs.…”

Structure and function of ribosomal RNA

“…However, since it is already clear that most if not all of the ribosomal proteins are capable of being cross-linked to their cognate RNA molecules in situ in the ribosomal subunits by one reagent or another, the question of determining the sites of cross-linking to individual proteins is now the dominant problem. A number of such crosslinking sites to various proteins on E. coli 23S RNA (Wower et al, 1981) or 16S RNA (Wower & Brimacombe, 1983) have been determined by using 2-iminothiolane as the cross-linking agent, and these results are included in Figs. 1 and 2, together with some more recent assignments obtained after cross-linking with 2-iminothiolane or nitrogen mustard (A. Kyriatsoulis, I. Wower & R. Brimacombe, unpublished results).…”

Long range RNA-RNA interactions in the 30 S ribosomal subunit ofE. coli

“…7 (31). f. Interaction may be stabilized by protein S20 (32,33). g. Interaction may be stabilized by proteins S4 and S12 (30,31,34).…”