RNA-protein cross-linking in Escherichia coli 30s ribosomal subunits: precise localisation of the nucleotide in 16s RNA which is coupled to protein S7 by ultraviolet irradiation

摘要

It is well established that when E. coil 30s ribosomal subunits are irradiated with ultraviolet light under mild conditions a specific cross-link is formed between protein S7 and the 16s RNA. Methodology is presented for the analysis of the single nucleotide residue concerned in this cross-link. Firstly, the identity of the ribonuclease T₁ octanucleotide attached to S7 is confirmed by a new method, which involves isolation and analysis of S7-polynucleotide complexes containing 30 – 40 nucleotides. Secondly, the isolated S7-octanucleotide complex is digested successively with ribonuclease A, proteinase K and ribonuclease T₂, and the nucleotides liberated are identified. The results show unambiguously that uridine residue number 1239 in the 16s RNA sequence is cross-linked to protein S7.