Cross‐linking between 16S ribosomal RNA and protein S4 in Escherichia coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate

Cytosine in nucleic acids can be modified by treatment with a mixture of bisulfite and hydrazine. The reaction is specific for single‐stranded regions of nucleic acids and the product is N⁴‐aminocytosine. Bromopyruvate has been used for alkylation of protein SH groups and through its 2‐oxo group it can form a hydrazone with N⁴‐aminocytosine. Escherichia coli ribosomal 30S subunits were treated with 1 M sodium bisulfite + 2 M hydrazine in the presence of 10 mM MgCl₂ at pH 7.0 and 37°C for 30 min. By this treatment, 2.4 cytosine residues/molecule 16S rRNA were derivatized into N⁴‐aminocytosines. ³⁵S‐labeled 30S subunits were modified in this way and then treated with 10 mM bromopyruvate at pH 8.0 and 37°C for 5 min. Analysis in sodium dodecyl sulfate/sucrose density gradient centrifugation showed co‐sedimentation of a part of the ³⁵S radioactivity with the RNA. The cosedimentation was dependent on both the bisulfite/hydrazine and the bromopyruvate treatments. The RNA‐protein complex was prepared from unlabeled 30S subunits. The protein portion was labeled with ¹²⁵I, the RNA portion was digested with nucleases, and then the hydrazone linkage between the protein and oligonucleotides was cleaved by treatment with 0.2 M HCl. The oligonucleotides formed were removed by dialysis and the protein was identified as S4 by two‐dimensional electrophoresis and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that the cysteinyl residue of protein S4 at position 31 from the N‐terminus is located close to a cytosine residue which is non‐base‐paired and easily accessible by the externally present bisulfite/hydrazine reagent.