Bacteroides expand the functional versatility of a universal transcription factor and transcribed DNA to program capsule diversity

Human gut Bacteroides species encode numerous (eight or more) tightly regulated capsular polysaccharides (CPS). Specialized paralogs of the universal transcription elongation factor NusG, called UpxY (Y), and an anti-Y UpxZ (Z) are encoded by the first two genes of each CPS operon. The Y-Z regulators combine with promoter inversions to limit CPS transcription to a single operon in most cells. Y enhances transcript elongation whereas Z inhibits noncognate Ys. How Y distinguishes among cognate CPS operons and how Z inhibits only noncognate Ys are unknown. Using in-vivo nascent-RNA sequencing and promoter-less in-vitro transcription (PIVoT), we establish that Y recognizes a paused RNA polymerase via sequences in both the exposed non-template DNA and the upstream duplex DNA. Y association is aided by novel ‘pause-then-escape’ nascent RNA hairpins. Z binds non-cognate Ys to directly inhibit Y association. This Y-Z hierarchical regulatory program allows Bacteroides to create CPS subpopulations for optimal fitness. Examination of nascent transcripts from a filtered culture of Bacteroides fragilis rpoC-3xFLAG

人体肠道拟杆菌属物种编码大量（至少8种）受严格调控的荚膜多糖（CPS, capsular polysaccharides）。每一个CPS操纵子的前两个基因分别编码通用转录延伸因子NusG的特化旁系同源蛋白UpxY（简称Y）与抗Y蛋白UpxZ（简称Z）。Y-Z调控因子与启动子倒位协同作用，使绝大多数细胞内的CPS转录仅局限于单个操纵子。Y可促进转录延伸，而Z则抑制非同源Y蛋白的功能。目前Y如何区分同源CPS操纵子，以及Z为何仅能靶向抑制非同源Y蛋白，仍是未解之谜。本研究借助体内新生RNA测序（in-vivo nascent-RNA sequencing）与无启动子体外转录（PIVoT, promoter-less in-vitro transcription）技术，证实Y可通过暴露的非模板DNA与上游双链DNA中的序列识别处于暂停状态的RNA聚合酶。Y的结合还受到新型“先暂停后逃逸”新生RNA发夹结构的辅助。Z可结合非同源Y蛋白，直接阻断Y的结合过程。这套Y-Z层级调控程序使得拟杆菌能够构建CPS亚群以实现最优适应性。本研究还对经过滤培养的脆弱拟杆菌（Bacteroides fragilis）rpoC-3xFLAG菌株的新生转录本进行了分析