RNA-protein cross-linking in Escherichia coli 50S ribosontal subunits; determination of sites on 23S RNA that are cross-linked to proteins L2, L4, L24 and L27 by treatment with 2-iminothiolane

摘要

RNA-protein cross-links were introduced into E. coli 50S ribosomal subunits by treatment with 2-iminothiolane followed by mild ultraviolet irradiation. After partial digestion of the RNA, the cross-linked RNA-protein complexes were separated by our recently published three-step procedure. In cases where this separation was inadequate, a further purification step was introduced, involving affinity chromatography with antibodies to the ribosomal 50S proteins. Analysis of the isolated complexes enabled four new cross-link sites on the 23S RNA to be identified, as well as re-confirming several previously established sites. The new sites are as follows: Protein L2 is cross-linked within an oligonucleotide at positions 1818–1823 in the 23S RNA, protein L4 within positions 320–325, protein L24 within positions 99–107, and protein L27 within positions 2320–2323.