Ribonucleic acid-protein cross-linking within the intact Escherichia coli ribosome, utilizing ethylene glycol bis[3-(2-ketobutyraldehyde) ether], a reversible, bifunctional reagent: synthesis and cross-linking within 30S and 50S subunits.
We have used the reversible, bifunctional reagent ethylene glycol bis[3-(2-ketobutyraldehyde) ether] to cross-link RNA to protein within intact ribosomal subunits from Escherichia coli. Here we describe the synthesis of this compound (termed bikethoxal) and demonstrate its ability to form covalent attachments between RNA and protein in the 5S RNA-L18 complex and within 30S and 50S ribosomal subunits. The reagent is a symmetrical dicarbonyl compound and reacts with guanine in single-stranded RNA and with arginine in protein. RNA-protein cross-links generated with this reagent are stable, as demonstrated by the comigration of 35S-labeled ribosomal proteins with ribosomal RNA on neutrally buffered sodium dodecyl sulfate (SDS)-agarose gels. However, the cross-linked product is unstable in mildly basic conditions, allowing the identification of the linked macromolecules by conventional techniques. The reagent is potentially capable of cross-linking any combination of single-stranded RNA, single-stranded DNA, or protein; it should prove a useful probe of the RNA-protein proximities within the E. coli ribosome, since the SDS-agarose gel system we describe provides a rapid method of optimizing this RNA--protein cross-linking reaction.
D000447:Aldehydes / Q000138:chemical synthesis / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012335:RNA, Ribosomal / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure
D000447:Aldehydes; D003432:Cross-Linking Reagents; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C029609:ribosomal protein L18; C039326:bikethoxal