Chen S M, Takiff H E, Barber A M, Dubois G C, Bardwell J C, Court D L
Molecular Control and Genetics Section, National Cancer Institute, Frederick, Maryland.
J Biol Chem. 1990 Feb 15;265(5):2888-95.
The synthesis rates of ribonuclease III (RNase III) and Era proteins are relatively low, and expression of the era gene is translationally coupled with expression of the rnc gene. Expression of both genes is negatively controlled by RNase III itself. We have constructed plasmids that overproduce RNase III and/or Era proteins under the control of the lambda PL promoter. A plasmid with the rnc gene under PL control expresses RNase III at levels greater than 40% of total cellular protein. Another plasmid with the era gene under PL control and a modified translation-initiation signal produces up to 80% of total cell protein as Era. Each protein has been purified using simple and rapid procedures. Purified RNase III protein specifically processes mRNA transcripts containing known RNase III sites. The purified Era protein binds GDP and GTP and has GTPase activity. Kinetic analysis shows that one molecule of GTP or GDP is bound/Era peptide with a Kd of 5.5 microM for GTP binding and 1.0 microM for GDP binding. The Km of the Era GTPase is 9.0 microM, and the maximum catalyzed rate of GTP hydrolyzed/min/mol of Era protein at 37 degrees C is 9.8 mmol.
核糖核酸酶III（RNase III）和Era蛋白的合成速率相对较低，并且era基因的表达与rnc基因的表达在翻译水平上偶联。这两个基因的表达均受到RNase III自身的负调控。我们构建了在λPL启动子控制下过量产生RNase III和/或Era蛋白的质粒。一个在PL控制下带有rnc基因的质粒表达的RNase III水平超过细胞总蛋白的40%。另一个在PL控制下带有era基因和修饰的翻译起始信号的质粒产生的Era蛋白高达细胞总蛋白的80%。每种蛋白都使用简单快速的方法进行了纯化。纯化的RNase III蛋白特异性地加工含有已知RNase III位点的mRNA转录本。纯化的Era蛋白结合GDP和GTP并具有GTP酶活性。动力学分析表明，一个GTP或GDP分子与Era肽结合，GTP结合的解离常数（Kd）为5.5微摩尔，GDP结合的解离常数为1.0微摩尔。Era GTP酶的米氏常数（Km）为9.0微摩尔，在37℃时，每摩尔Era蛋白每分钟催化GTP水解的最大速率为9.8毫摩尔。
