New RNA-protein crosslinks in domains 1 and 2 of E. coli 30S ribosomal subunits obtained by means of an intrinsic photoaffinity probe.
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 366 nm photoactivation of s4U. We observe the systematic and characteristic formation of 30S 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 S3, S4, S5, S7, S9, S18, S20 and S21. Several crosslinks are compatible with previously published sites for proteins S5, S18, S20 and S21; others for proteins S3, S4, S7, S9, S18 correspond necessarily to new sites.
Hajnsdorf
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Institut Jacques Monod, CNRS-Université Paris VII, France.
Favre
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Expert-Bezançon
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D002460:Cell Line; D003432:Cross-Linking Reagents / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010957:Plasmids; D012335:RNA, Ribosomal / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D015183:Restriction Mapping; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013891:Thiouridine / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; C040469:ribosomal protein S30; D013891:Thiouridine; D008274:Magnesium