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

Noriko NITTA, Osamu KUGE, Seiko YUI, Kazuo NEGISHI, Hikoya HAYATSU

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1 Citation (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)427-432
Number of pages6
JournalEuropean Journal of Biochemistry
Volume157
Issue number2
DOIs
Publication statusPublished - Jun 1986
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry

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