Su S L, Dubnau D
Department of Microbiology, Public Health Research Institute, New York, New York 10016.
Biochemistry. 1990 Jun 26;29(25):6033-42. doi: 10.1021/bi00477a022.
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 x 10(-9) M at 37 degrees C. Kinetic studies of complex formation and dissociation revealed that the kon and koff were 4 x 10(6) M-1 s-1 and 6.8 x 10(-2) s-1 respectively at 16 degrees C. Equilibrium competition experiments showed that the enzyme has varying affinities for a variety of nucleic acids in the order 23S rRNA greater than 16S rRNA greater than M13 DNA, f2 RNA greater than 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 (delta H = +6.2 kcal mol-1) and was driven by entropy (delta S = +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 approximately 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.
ermC 23S rRNA甲基转移酶可使枯草芽孢杆菌23S rRNA中的腺嘌呤2085发生二甲基化，并且还通过自身的翻译抑制来调节其自身的合成。我们已经对ermC'甲基转移酶与23S rRNA的结合进行了表征。该蛋白质与ermC产物仅在五个氨基酸残基上有所不同，因其具有更高的稳定性和易于分离的特点而被选作研究对象。采用滤膜结合试验研究了在不存在甲基化的情况下结合的物理特性。发现结合的解离平衡常数在37℃时为4×10⁻⁹ M。复合物形成和解离的动力学研究表明，在16℃时，结合速率常数kon和解离速率常数koff分别为4×10⁶ M⁻¹ s⁻¹和6.8×10⁻² s⁻¹。平衡竞争实验表明，该酶对多种核酸具有不同的亲和力，顺序为23S rRNA＞16S rRNA＞M13 DNA，f2 RNA＞tRNA。甲基化的终产物之一，即甲基化的23S rRNA，对ermC'甲基转移酶的亲和力与未甲基化的23S rRNA相似。AdoMet的存在对与23S rRNA的结合亲和力和相互作用动力学没有明显影响。ermC'甲基转移酶与23S rRNA的结合具有不利的范特霍夫焓（ΔH = +6.2 kcal mol⁻¹），并且由熵驱动（ΔS = +56.2 cal mol⁻¹ deg⁻¹）。两种配体之间的相互作用最多涉及两到三个离子对，非静电相互作用约占结合能的85%。通过用硫酸二甲酯进行探测，研究了ermC'甲基转移酶对23S rRNA的依赖性保护作用，以此来探究相互作用的结构方面。在rRNA结构域V的中央环附近以及甲基化位点周围检测到了一个保护区域。
