Indian Institute of Science Education and Research Mohali, Sector 81, SAS Nagar 140306, Punjab, India.
Centre for DNA Fingerprinting and Diagnostics, Uppal Road, Hyderabad 500039, Telengana, India.
Nucleic Acids Res. 2024 Nov 11;52(20):12438-12455. doi: 10.1093/nar/gkae839.
In many bacteria, the essential factors Rho and NusG mediate termination of synthesis of nascent transcripts (including antisense RNAs) that are not being simultaneously translated. It has been proposed that in Rho's absence toxic RNA-DNA hybrids (R-loops) may be generated from nascent untranslated transcripts, and genome-wide mapping studies in Escherichia coli have identified putative loci of R-loop formation from more than 100 endogenous antisense transcripts that are synthesized only in a Rho-deficient strain. Here we provide evidence that engineered expression in wild-type E. coli of several such individual antisense regions on a plasmid or the chromosome generates R-loops that, in an RNase H-modulated manner, serve to disrupt genome integrity. Rho inhibition was associated with increased prevalence of antisense R-loops also in Xanthomonas oryzae pv. oryzae and Caulobacter crescentus. Our results confirm the essential role of Rho in several bacterial genera for prevention of toxic R-loops from pervasive yet cryptic endogenous antisense transcripts. Engineered antisense R-looped regions may be useful for studies on both site-specific impediments to bacterial chromosomal replication and the mechanisms of their resolution.
在许多细菌中,必需因子 Rho 和 NusG 介导新生转录物(包括反义 RNA)合成的终止,这些转录物没有被同时翻译。有人提出,在 Rho 缺失的情况下,可能会从新生的未翻译转录物中产生有毒的 RNA-DNA 杂交体(R 环),而在大肠杆菌中的全基因组映射研究已经鉴定出了 100 多个内源性反义转录本的潜在 R 环形成部位,这些转录本仅在 Rho 缺陷型菌株中合成。在这里,我们提供的证据表明,在野生型大肠杆菌中,通过质粒或染色体上的工程表达,几个这样的单个反义区域会产生 R 环,这些 R 环以 RNase H 调节的方式,破坏基因组的完整性。Rho 抑制也与稻黄单胞菌 pv。oryzae 和新月柄杆菌中反义 R 环的普遍性增加有关。我们的研究结果证实了 Rho 在几个细菌属中预防有毒 R 环的重要作用,这些 R 环来自普遍存在但隐匿的内源性反义转录物。工程化的反义 R 环区域可能对研究细菌染色体复制的特异性障碍及其解决机制有用。