Zou L L, Richardson J P
Department of Chemistry, Indiana University, Bloomington 47405.
J Biol Chem. 1991 Jun 5;266(16):10201-9.
The efficiencies of rho action as a termination factor during transcription in vitro of several DNA templates were determined as a function of the concentration and type of electrolyte ions. The termination efficiencies with lambda-tR1 and the promoter proximal lacZ intragenic terminators were significantly higher with 0.1-0.2 M potassium glutamate as the major electrolyte than with the optimal concentrations of KCl (approximately 0.05 M) or potassium acetate (approximately 0.15 M). Similar high efficiencies were obtained with salts of other acidic amino acids but not with a salt of N-acetylglutamic acid or with a mixture of 0.15 M potassium acetate and 0.15 M glycine, and termination was inhibited completely when 0.12 M KCl was present along with 0.12 M potassium glutamate. The salts that give high termination efficiencies have two properties in common; they consist of anions that are also zwitterions, and they are weak chelators of Mg2+ ions. The increase in termination efficiency with potassium glutamate can be ascribed mainly to a facilitation of the reactions of rho with RNA that are coupled to ATP hydrolysis, as the rate of ATP hydrolysis with isolated transcripts as cofactors was about five times higher with 0.15 M potassium glutamate than with 0.05 M KCl, whereas the rates of chain elongation, the general stability of the transcription complexes, and the binding affinity of rho with the transcripts were all very similar under the two conditions. Further analysis revealed that the activation of ATP hydrolysis is an outcome of a shift in the optimum magnesium salt concentration from 0.5 mM with 0.05 M KCl to 4 mM with 0.15 M potassium glutamate. Since glutamate is a relatively weak counterion for cationic groups in proteins, potassium glutamate can be used at 0.15 M without inhibiting the binding of rho to RNA. At that concentration, it serves to buffer the level of free Mg2+ available to stabilize RNA secondary structures that are known to impede rho action on RNA. The two special properties of glutamate together create conditions that allow rho to terminate transcription in vitro at an efficiency that matches the in vivo efficiency with use of a physiological level of K+ ions.
在体外转录过程中，测定了rho作为终止因子对几种DNA模板发挥作用的效率，该效率是电解质离子浓度和类型的函数。以0.1 - 0.2M谷氨酸钾作为主要电解质时，λ-tR1和启动子近端lacZ基因内终止子的终止效率显著高于KCl（约0.05M）或醋酸钾（约0.15M）的最佳浓度时的终止效率。其他酸性氨基酸的盐也能获得类似的高效率，但N - 乙酰谷氨酸盐或0.15M醋酸钾与0.15M甘氨酸的混合物则不能，当0.12M KCl与0.12M谷氨酸钾同时存在时，终止完全受到抑制。能产生高终止效率的盐有两个共同特性；它们由也是两性离子的阴离子组成，并且是Mg2 +离子的弱螯合剂。谷氨酸钾导致终止效率增加主要可归因于促进了rho与RNA的反应，这些反应与ATP水解偶联，因为以分离的转录本作为辅因子时，0.15M谷氨酸钾条件下的ATP水解速率比0.05M KCl时高约五倍，而在这两种条件下，链延伸速率、转录复合物的总体稳定性以及rho与转录本的结合亲和力都非常相似。进一步分析表明，ATP水解的激活是最佳镁盐浓度从0.05M KCl时的0.5mM转变为0.15M谷氨酸钾时的4mM的结果。由于谷氨酸是蛋白质中阳离子基团相对较弱的抗衡离子，谷氨酸钾可以在0.15M的浓度下使用而不抑制rho与RNA的结合。在该浓度下，它用于缓冲可用于稳定已知会阻碍rho对RNA作用的RNA二级结构的游离Mg2 +水平。谷氨酸的这两个特殊特性共同创造了条件，使得rho在体外以与使用生理水平K +离子时的体内效率相匹配的效率终止转录。
