Project Summary/Abstract RNA polymerase (RNAP) pausing and termination are important components of gene expression in all organisms. NusA and NusG are two general transcription elongation factors that are capable of stimulating pausing and termination in bacteria. Pausing allows synchronization of the position of RNAP with RNA folding and/or regulatory factor binding. Using a method that combines nascent elongating transcript sequencing with RNase I digestion (RNET-seq), it was determined that NusG-dependent pausing occurs at 1,600 sites throughout the B. subtilis genome. ~25% of these pause sites are in 5'UTRs, and the role that several of these 5'UTR pauses have in regulating downstream gene expression will be examined. The other 75% of the pause sites are in open reading frames and the possibility that some of these pauses are involved in maintaining coupling of transcription and translation will be tested. The structural basis for NusG-dependent pausing will also be investigated using cryo-electron microscopy (Cryo-EM). The in vivo roles of NusA and NusG in pausing have not been explored in E. coli because both proteins are essential. The recent ability to deplete NusA and NusG will be exploited to examine NusA-dependent and NusG-dependent pausing in E. coli using RNET-seq. Promoter proximal pausing is a distinct pausing mechanism that is mediated by σ factor interaction with -10 promoter elements or -10-like sequences in the DNA. These backtracked pauses are relieved by Gre factors that stimulate the RNA cleavage activity of RNAP. Following cleavage, the RNA 3' end becomes properly aligned in the active site such that elongation can resume. The prevalence of σA-dependent promoter proximal pausing in B. subtilis, as well as the role that GreA plays in this process, will be investigated using RNET-seq. Intrinsic and Rho-dependent termination are generally thought to occur via two distinct and non-overlapping mechanisms. Intrinsic terminators consist of an RNA hairpin followed by a U-rich tract. Using a 3' end-mapping strategy (Term-seq) it was shown that that NusA and NusG function as general intrinsic termination factors in B. subtilis. NusA-dependent terminators have weak RNA hairpins and/or poor U-tracts, whereas NusG-dependent terminators require NusG-dependent pausing to provide sufficient time for folding of hairpins with weak A-U base pairs at the bottom of the stem. In Rho-dependent termination, Rho promotes transcript release when it catches up to paused RNAP. E. coli NusG participates in some Rho-dependent termination events by serving as a bridge between RNAP and Rho. Of particular interest, B. subtilis Rho functions as a third intrinsic termination factor, in stark contrast to the accepted view that Rho only participates in canonical Rho-dependent termination. The mechanism of Rho-dependent intrinsic termination, and the potential role of NusA or NusG in this process, will be explored. In addition, Term-seq will be used to perfor...