Harinarayanan R, Gowrishankar J
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.
J Mol Biol. 2003 Sep 5;332(1):31-46. doi: 10.1016/s0022-2836(03)00753-8.
Two Escherichia coli genes, rnhA and recG, encode products that disrupt R-loops by hydrolysis and unwinding, respectively. It is known that the propensity for R-loop formation in vivo is increased during growth at 21 degrees C. We have identified several links between rnhA, recG, and R-loop-dependent plasmid replication on the one hand, and genes rho and nusG involved in factor-dependent transcription termination on the other. A novel nusG-G146D mutation phenocopied a rho-A243E mutation in conferring global deficiency in transcription termination, and both mutants were killed at 21 degrees C following overexpression of rnhA(+). Mutant combinations rnhA-nusG or recG-rho were synthetically lethal at 21 degrees C, with the former being suppressed by recG(+) overexpression. rho and nusG mutants were killed following transformation with plasmids such as pACYC184 or pUC19 (which have R-loop replication intermediates) even at 30 degrees C or 37 degrees C, and the lethality was correlated with greatly increased content of supercoiled monomer species of these and other co-resident R-loop-dependent plasmids. Plasmid-mediated lethality in the mutants was suppressed by overexpression of rnhA(+) or recG(+). Two additional categories of trans-acting suppressors of the plasmid-mediated lethality were identified whose primary effects were, respectively, a reduction in plasmid copy number even in the wild-type strain, and a restoration of the proficiency of in vivo transcription termination in the nusG and rho mutant strains. The former category of suppressors included rom(+), and mutations in rpoB(Q513L), pcnB, and polA, whereas the latter included a mutation in rho (R221C) and several non-null mutations (E74K, L26P, and delta64-137) in the gene encoding the nucleoid protein H-NS. We propose that an increased occurrence of chromosomal R-loops in the rho and nusG mutants leads to titration of a cyloplasmic host factor(s) that negatively modulates the stability of plasmid R-loop replication intermediates and consequently to runaway plasmid replication.
两个大肠杆菌基因rnhA和recG分别编码通过水解和解旋破坏R环的产物。已知在21摄氏度生长期间，体内R环形成的倾向会增加。一方面，我们已经确定了rnhA、recG与R环依赖性质粒复制之间的若干联系，另一方面，也确定了与因子依赖性转录终止相关的rho和nusG基因之间的若干联系。一种新的nusG - G146D突变在转录终止方面表现出与rho - A243E突变相似的全局缺陷，并且在rnhA(+)过表达后，这两种突变体在21摄氏度时都会死亡。rnhA - nusG或recG - rho突变组合在21摄氏度时具有合成致死性，前者可通过recG(+)过表达得到抑制。即使在30摄氏度或37摄氏度时，用诸如pACYC184或pUC19（具有R环复制中间体）的质粒转化rho和nusG突变体后，它们也会死亡，并且致死性与这些以及其他共驻留的R环依赖性质粒的超螺旋单体种类含量大幅增加相关。rnhA(+)或recG(+)的过表达可抑制突变体中质粒介导的致死性。还鉴定出了另外两类质粒介导致死性的反式作用抑制子，其主要作用分别是即使在野生型菌株中也降低质粒拷贝数，以及恢复nusG和rho突变体菌株体内转录终止的能力。前一类抑制子包括rom(+)以及rpoB(Q513L)、pcnB和polA中的突变，而后者包括rho中的一个突变（R221C）以及编码类核蛋白H - NS的基因中的几个非无效突变（E74K、L26P和delta64 - 137）。我们提出，rho和nusG突变体中染色体R环的发生率增加导致一种细胞质宿主因子被滴定，该因子对质粒R环复制中间体的稳定性产生负调节作用，从而导致质粒复制失控。
