Binding of Bacillus subtilis ermC′ Methyltransferase to 23S rRNA

Sai L. Su, David Dubnau

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Abstract

ermC 23S rRNA methyltransferase dimethylates adenine 2085 in Bacillus subtilis 23S rRNA and also regulates its own synthesis by autogenous translational repression. We have characterized the binding of ermC′ methyltransferase to 23S rRNA. This protein differs in only five amino acid residues from the ermC product and was chosen for study because of its greater stability and ease of isolation. A filter binding assay was used to study the physical aspects of binding in the absence of methylation. The dissociation equilibrium constant of the binding was found to be 4 × 10−9 M at 37°C. Kinetic studies of complex formation and dissociation revealed that the kon and koff were 4 × 106 M−1 s−1 and 6.8 × 10−2 s−1 respectively at 16°C. Equilibrium competition experiments showed that the enzyme has varying affinities for a variety of nucleic acids in the order 23S rRNA > 16S rRNA > M13 DNA, f2 RNA > tRNA. One of the end products of methylation, methylated 23S rRNA, had an affinity for the ermC′ methyltransferase similar to that of unmethylated 23S rRNA. The binding affinity to 23S rRNA and the kinetics of the interaction were not detectably affected by the presence of AdoMet. The binding of ermC′ methyltransferase to 23S rRNA had an unfavorable van’t Hoff enthalpy (∆H = +6.2 kcal mol−1) and was driven by entropy (AS = +56.2 cal mol−1 deg−1). The interaction between the two ligands involved at most two to three ionic pairings, and nonelectrostatic interactions contributed ~85% of the binding energy. The structural aspect of the interaction was investigated by probing with dimethyl sulfate, for ermC’ methyltransferase dependent protection of 23S rRNA. A region of protection was detected, in the vicinity of the central loop of rRNA domain V and surrounding the site of methylation.

Original languageEnglish (US)
Pages (from-to)6033-6042
Number of pages10
JournalBiochemistry
Volume29
Issue number25
DOIs
StatePublished - Jun 1 1990

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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