New RNA-protein crosslinks in domains 1 and 2 of E.coli 30S ribosomal subunits obtained by means of an intrinsic photoaffinity probe (original) (raw)

1989, Nucleic Acids Research

Functionally active 70S ribosomes containing 4-thiouridine (s4U in place of uridine were prepared by a formerly described in vivo substitution method. Proteins were crosslinked to RNA by 366nm photoactivation of s"U. We observe the systematic and caracteristic formation of 305 dimers; they were eliminated for analysis of RNA-protein crosslinks. M13 probes containing rDNA inserts complementary to domains 1 and 2 of 16S RNA from the 5'end up to nucleotide 868 were used to select contiguous or overlapping RNA sections. The proteins covalently crosslinked to each RNA section were identified as 53. 54, 55. 57, 59, S18, S20 and S21. Several crosslinks are compatible with previously published sites for proteins S5, 518, 520 and 521 ; others for proteins 53, 54, 57, 59, 518 correspond necessarily to new sites. 1475 Nucleic Acids Research Volume 17 Number 4 1989 Nucleic Acids Research covalent crosslinked complexes. As a general and systematic approach to obtain low resolution mapping of crosslinked 305 proteins on 165 RNA. We have used single stranded Mi 3 DNA probes (ssDNA) containing rDNA inserts complementary to 16S RNA sections. These probes were hybridized to 1 6S RNA-protein complexes and RNase Ti hydrolysis was used to eliminate the non selected regions. The proteins covalently linked to the RNA sections protected by the complementary rDNA were identified by two-dimensional gel analysis. This method was previously described in details for domains 3 and 4 (12-14). We report here the use of 8 other M13 ssDNA probes to identify proteins crosslinked to sections between nucleotides 1 and 869, covering domains 1 and 2. We have found several RNA-protein crosslinks which are compatible with previous results and a few others that are necessarily new sites. MATERIAL AND METHODS. Material and Enzymes. 4-thiouridine was from Sigma. (1251) and the T4 DNA ligase were from Amersham (England). Carrier free (H932P04) was from CEA (France). The Klenow fragment or DNA polymerase was from Boehringer and RNAse T 1, RNase T2 from Sankyo. Buffers. Buffer A: 10 mM triethanolamine / HCl pH 7.5, 1 mM Mg AC2, 50 mM KCl Buffer B: 1OmM triethanolamine / HC1 pH 7.5; 0.1% SDS, 100 mM LICI. Buffer SCE I X: 15mM sodium citrate pH 7, 150mM NaCl, 1OmM Na2 EDTA. Buffer TBE 1X: 90mM Tris, 90mM borate , 2.5mM Na2 EDTA pH 8.3. Cell strains and media used for ribosome DreDaration.