Precise localisation of three intra-RNA cross-links in 23S RNA, and aone in 5S RNA, induced by treatment ofEscherichia coli50S ribosomal subunits withbis-(2-chloroethtyl)-methylamine

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Abstract
Treatment of E. coli 50S ribosomal subsunits with low doses of bis-(2-chloroethyl)-methylamine (“nitrogen mustard”) leads to formation of a number of intra-RNA and RNA-protein cross-links. After partial digestion of the cross-linked subunits with cobra venom nuclease, followed by destruction of the protein moiety with proteinase K, complexes containing the intra-RNA cross-links were isolated by two-dimensional gel electrophoresis. The individual complexes were subjected to oligonucleotide analysis,either directly or after a second partial digestion procedure using ribonuclease T1, and the cross-link sites determined. In 235 RNA, the cross-links found were between bases 763 and 1567, 1210 and 1236, 1482 and 1501; in 5S RNA, base 69 was cross-linked to base 107. The significance of these cross-links in relation to the three-dimensional organization of the ribosomal RNA is discussed.