Chemical cross-linking of protein to RNA within intact ribosomal subunits from Escherichia coli.
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.
D002074:Butanones / Q000494:pharmacology; D004586:Electrophoresis; D004926:Escherichia coli; D008968:Molecular Conformation; D009588:Nitrogen Mustard Compounds / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D011422:Propionates / Q000494:pharmacology; D012329:RNA, Bacterial / Q000032:analysis / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000032:analysis / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000032:analysis / Q000378:metabolism*
D002074:Butanones; D009588:Nitrogen Mustard Compounds; D011422:Propionates; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C016280:bromo-ketone reagent