Chemical cross-linking of protein to RNA within intact ribosomal subunits from Escherichia coli

Abstract

Bifunctional reagents, namely bis-(2-chloroethyl)-amine ("nitrogen mustard") and activated esters of 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid ("bromo-ketone reagent") are used to cross-linked protein to RNA within intact ribosomal subunits. The cross-linked proteins are analysed on two different two-dimensional gel electrophoresis sytems, and the existence of a stable cross-linkage is demonstrated by isolating cross-linked protein-oligonucleotide complexes from subunits containing 32P-labelled RNA. Proteins S3, S4, S5, S9/S11 and S13 from the 30S subunit, and proteins L1 and L2 from the 50S subunit were cross-linked to RNA by the nitrogen mustard, together with a number of other so far unresolved proteins. Correspondingly S3, S4, S7, S9/S11 and L12 were cross-linked by the bromoketone reagent, although in lower yield. The reagents should prove useful topographical studies on ribosomal subunits, and arguments are presented favouring the use of non-cleavable and relatively non-specific RNA-protein cross-linking reagents for such studies.

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

“…For this purpose, 2 A260 units of unlabelled 30S subunits were added, and the RNA moiety was fully digested by incubation with ribonucleases A and Tl, followed by ethanol precipitation to remove oligonucleotides, as described in ref. 19. The proteins were then separated on the two-dimensional system of Geyl et al (17), the gels being stained and the protein spots analysed for 14C-radioactivity, exactly as described above.…”

The use of 2-iminothiolane as an RNA-protein cross-linking agent in Escherichia coli ribosomes, and the localisation on 23S RNA of sites cross-linked to proteins L4, L6, L21, L23, L27 and L29

“…(a) To determine the extent of reaction, aliquots of doublelabelled 30S or 50S subunits (1-2 A260 units) were used. After cross-linking as described above, the reaction mixtures were applied to 3-8% polyacrylamide gels containing dodecyl sulphate (1,2,6). The percentage of RNA-protein cross-linking was determined from the amount of 14C-protein co-migrating with the peak of 3H-RNA in the gel.…”

“…3 A260 units of double-labelled subunit. After crosslinking, the reaction mixtures were applied to sucrose gradients in dodecyl sulphate (1,2,6). The peaks of RNA plus RNA-protein cross-linked complex isolated from the gradients were in each case subjected to digestion with ribonucleases A and T1 as previously described (2).…”

“…Our objective has been to introduce such cross-links in situ into the ribosomal subunits, and then to determine the precise sites of cross-linking on both protein and RNA, in order to build up a detailed topographical map of RNA-protein contacts and neighbourhoods within the particles. Up to now this research has been pursued in two directions, firstly the search for suitable bifunctional cross-linking reagents (1,2,4,6) and secondly the development of techniques for the analysis of the cross-link sites (3,5,7).…”

“…A number of symmetrical bifunctional reagents were found which could be used as RNA-protein cross-linking agents (2,4, and cf. 8), but only recently has a genuine hetero-bifunctional reagent been developed for the ribosome, namely p-azido phenyl-acetic imidoester (APAI, 6).…”