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 p romoter-less in v itr o 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.