Transcription elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native-elongating-transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G−10Y−1G+1 (where −1 corresponds to the position of the RNA 3′ end). We demonstrate that sequence-specific interactions between RNA polymerase core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a post-translocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.

SUMMARY We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 47 (~16,000) bar-coded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields ("massively systematic transcript end readout," MASTER). Using MASTER, we define full inventories of transcription start sites ("TSSomes") of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo, we define the TSS-region DNA-sequence determinants for TSS selection, reiterative initiation ("slippage synthesis"), and transcript yield, and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion ("scrunching") and transcription-bubble contraction ("anti-scrunching").

SUMMARY It is often presumed that, in vivo, the initiation of RNA synthesis by DNA-dependent RNA polymerases occurs using NTPs alone. Here, using the model Gram-negative bacterium Pseudomonas aeruginosa, we demonstrate that depletion of the small-RNA-specific exonuclease, Oligoribonuclease, causes the accumulation of 2- to ~4-nt RNAs, “nanoRNAs”, which serve as primers for transcription initiation at a significant fraction of promoters. Widespread use of nanoRNAs to prime transcription initiation is coupled with global alterations in gene expression. Our results, obtained under conditions in which the concentration of nanoRNAs is artificially elevated, establish that small RNAs can be used to initiate transcription in vivo, challenging the idea that all cellular transcription occurs using NTPs alone. Our findings further suggest that nanoRNAs could represent a distinct class of functional small RNAs that can affect gene expression through direct incorporation into a target RNA transcript rather than through a traditional antisense-based mechanism.

The Shigella outer membrane protease IcsP removes the actin assembly protein IcsA from the bacterial surface, and consequently modulates Shigella actin-based motility and cell-to-cell spread. Here, we demonstrate that IcsP expression is undetectable in mutants lacking either of two transcriptional activators, VirF and VirB. In wild-type Shigella spp., virB expression is entirely dependent on VirF; therefore, to circumvent this regulatory cascade, we independently expressed VirF or VirB in Shigella strains lacking both activators and measured both IcsP levels and transcription from the icsP promoter. Our results show that VirB significantly enhanced icsP transcription, even in the absence of VirF. In contrast, when VirF was induced in the absence of VirB, VirF had variable effects. The regulation of icsP is distinctly different from the regulation of the gene encoding its major substrate, icsA, which is activated by VirF and not VirB. We propose that the different pathways regulating icsA and icsP may be critical to the modulation of IcsA-mediated actin-based motility by IcsP.Shigella spp., gram-negative bacterial pathogens cause severe and bloody diarrhea in their human hosts by invading and spreading through the colonic epithelium. Shigella movement within the host cell cytoplasm is dependent on the ability of the bacterium to recruit host cell actin to its surface to form an "actin tail," which propels the bacterium from one cell to another (5,16,29). Actin tail assembly is mediated by a single bacterial protein, IcsA, which is found on the outer surface at one pole of the bacterium (17). This asymmetric localization of IcsA ensures that actin assembly occurs in a directional manner. In its mature form, IcsA is comprised of two domains: the ␣ domain (residues 53 to 758) contains the determinant for actin assembly (14) and extends from the bacterial surface into the extracellular environment, whereas the ␤ domain (residues 759 to 1102) is embedded in the outer membrane (33). The amount of IcsA ␣ domain exposed on the bacterial surface correlates with the efficiency of actin tail formation in the cytoplasm of infected cells (21).IcsP, an outer membrane protease of Shigella, cleaves IcsA between Arg 758 and Arg 759 , removing the entire IcsA ␣ domain from the bacterial surface (8, 13, 15a, 31). Overexpression of IcsP leads to complete removal of the IcsA ␣ domain from the bacterial cell surface (32), whereas genetic disruption of icsP increases the total amount of cell associated IcsA ␣ domain, leading to an increase in the rate of actin-based movement of Shigella (31). Although IcsP is not required for polar localization of IcsA (6,28), it contributes to the maintenance of a tight polar cap of IcsA on the bacterial surface (31). Furthermore, as Shigella enter stationary phase, the amount of cell-associated IcsA ␣ domain decreases dramatically, an effect due at least in part to IcsP (18,32).These data demonstrate that IcsP plays an important role in modulating the amount of the IcsA ␣ domain present on the bacter...

During transcription initiation in vitro, RNA polymerase can engage in abortive initiation-the synthesis and release of short, 2 to 15 nucleotide, RNA transcripts-prior to productive initiation. It has not been known whether abortive initiation occurs in vivo. Using hybridization with locked nucleic acid probes, we directly detect abortive transcripts in vivo and thereby show that abortive initiation occurs in vivo. We further show that abortive initiation in vivo is a determinant of promoter strength, a determinant of RNA polymerase function, and a target of regulation by transcription factor GreA. Abortive transcripts may have functional, physiologically important roles in regulating gene expression in vivo.During transcription, RNA polymerase (RNAP) synthesizes the first ∼8-15 nucleotides (nt) of RNA as an RNAP-promoter initial transcribing complex (1-3) [using a "scrunching" mechanism (4)]. Upon synthesis of an RNA transcript of a threshold length of ∼8-15 nt, RNAP breaks its interactions with promoter DNA, escapes the promoter, and enters into processive synthesis of RNA as an RNAP-DNA transcription elongation complex (1-3) [using a "stepping" mechanism (5)]. In transcription reactions in vitro, the RNAP-promoter initial transcribing complex can engage in tens to hundreds of cycles of synthesis and release of short RNA transcripts ("abortive initiation") (1-3,6-8). Abortive initiation competes with productive initiation in vitro and, as such, is a critical determinant of promoter strength and a target of transcription regulation in vitro (1-3,7-13). It has been proposed that abortive initiation likewise occurs in vivo (6,8). In support of this proposal, factors that affect yields of full-length transcripts in vitro through effects on abortive initiation likewise affect yields of full-length transcripts in vivo (9-14). However, no direct evidence that abortive initiation occurs in vivo has been presented.The bacteriophage T5 N25 promoter and its derivative N25anti are classic model systems for the study of abortive initiation and promoter escape (9,(11)(12)(13)(14). N25 and N25anti differ only in their initial transcribed sequences (positions +3 to +20) but exhibit radically different characteristics in vitro with respect to the abortive:productive ratio (APR; 40 for N25; ∼300 for N25anti), the maximum size of abortive transcripts (10 nt for N25; 15 nt for N25anti), and the kinetics of promoter escape (k clear ∼1.7 per min for N25; ∼0.06 per min for N25anti).To determine whether abortive initiation occurs in vivo, we sought to detect abortive transcripts generated during transcription from a plasmid-borne copy of N25anti in Escherichia coli, using locked nucleic acid probes developed for hybridization-based detection of microRNAs (15,16). We reasoned that the relatively high APR of N25anti would facilitate accumulation of detectable quantities of abortive transcripts and that the relatively high maximum size of