KorB switching from DNA-sliding clamp to repressor mediates long-range gene silencing in a multi-drug resistance plasmid

Examples of long-range gene regulation in bacteria are rare and generally thought to involve DNA looping. Here, using a combination of biophysical approaches including X-ray crystallography and single-molecule analysis for the KorB-KorA system in Escherichia coli, we show that long-range gene silencing on the plasmid RK2, a source of multi-drug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We show that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters up to 1.5 kb away. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed clamp state. DNA-bound KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Together, our findings explain the mechanistic basis for KorB role switching from a DNA-sliding clamp to a co-repressor, and provide an alternative mechanism for long-range regulation of gene expression in bacteria.

细菌中远距离基因调控的实例较为罕见，且既往普遍认为此类调控依赖DNA环化机制。本研究针对大肠杆菌（Escherichia coli）中的KorB-KorA系统，结合X射线晶体学（X-ray crystallography）与单分子分析（single-molecule analysis）等生物物理手段，证实了质粒RK2（plasmid RK2）上的远距离基因沉默现象——该质粒是多种革兰氏阴性菌（Gram-negative bacteria）多药耐药性（multi-drug resistance）的重要来源——由DNA滑动夹（DNA-sliding clamp）KorB与夹锁定蛋白（clamp-locking protein）KorA协同完成。研究表明，KorB属于CTP酶夹（CTPase clamp），能够捕获DNA并沿其滑动，抵达最远可达1.5 kb的远端靶启动子。我们解析了KorB-KorA-DNA三元复合物的晶体结构，揭示KorA可将KorB锁定为闭合夹状态。结合于DNA的KorA可通过在靶启动子处阻滞KorB滑动、阻断RNA聚合酶全酶（RNA polymerase holoenzymes）结合，从而介导转录抑制。综上，本研究阐明了KorB从DNA滑动夹转变为协同阻遏蛋白（co-repressor）的机制基础，并为细菌基因表达的远距离调控提供了一种全新的替代机制。