Jain Vikas, Saleem-Batcha Raspudin, China Arnab, Chatterji Dipankar
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Protein Sci. 2006 Jun;15(6):1449-64. doi: 10.1110/ps.062117006.
Latency in Mycobacterium tuberculosis poses a barrier in its complete eradication. Overexpression of certain genes is one of the factors that help these bacilli survive inside the host during latency. Among these genes, rel, which leads to the expression of Rel protein, plays an important role by synthesizing the signaling molecule ppGpp using GDP and ATP as substrates, thereby changing bacterial physiology. In Gram-negative bacteria, the protein is thought to be activated in vivo in the presence of ribosome by sensing uncharged tRNA. In the present report, we show that Rel protein from Mycobacterium smegmatis, which is highly homologous to M. tuberculosis Rel, is functional even in the absence of ribosome and uncharged tRNA. From the experiments presented here, it appears that the activity of Rel(Msm) is regulated by the domains present at the C terminus, as the deletion of these domains results in higher synthesis activity, with little change in hydrolysis of ppGpp. However, in the presence of tRNA, though the synthesis activity of the full-length protein increases to a certain extent, the hydrolysis activity undergoes drastic reduction. Full-length Rel undergoes multimerization involving interchain disulfide bonds. The synthesis of pppGpp by the full-length protein is enhanced in the reduced environment in vitro, whereas the hydrolysis activity does not change significantly. Mutations of cysteines to serines result in monomerization with a simultaneous increase in the synthesis activity. Finally, it has been possible to identify the unique cysteine, of six present in Rel, required for tRNA-mediated synthesis of ppGpp.
结核分枝杆菌的潜伏期是其彻底根除的一个障碍。某些基因的过表达是帮助这些杆菌在潜伏期内在宿主体内存活的因素之一。在这些基因中，rel基因可导致Rel蛋白的表达，它通过以GDP和ATP为底物合成信号分子ppGpp发挥重要作用，从而改变细菌生理状态。在革兰氏阴性菌中，该蛋白被认为在核糖体存在的情况下通过感知无电荷的tRNA在体内被激活。在本报告中，我们表明耻垢分枝杆菌的Rel蛋白与结核分枝杆菌的Rel高度同源，即使在没有核糖体和无电荷tRNA的情况下也具有功能。从这里展示的实验来看，Rel（耻垢分枝杆菌）的活性似乎受C末端结构域的调控，因为这些结构域的缺失会导致更高的合成活性，而ppGpp的水解变化不大。然而，在tRNA存在的情况下，尽管全长蛋白的合成活性在一定程度上增加，但水解活性却大幅降低。全长Rel会发生涉及链间二硫键的多聚化。在体外还原环境中，全长蛋白对pppGpp的合成增强，而水解活性没有显著变化。半胱氨酸突变为丝氨酸会导致单体化，同时合成活性增加。最后，已经能够确定Rel中六个半胱氨酸中tRNA介导的ppGpp合成所需的独特半胱氨酸。
