Seth R. Goldman

Direct Detection of Abortive RNA Transcripts in Vivo

Identifying Abortive Initiation During transcription initiation in vitro, the RNA polymerase enzyme typically engages in cycles of synthesis and release of short RNA transcripts (“abortive initiation”) before breaking interactions with promoter DNA and beginning transcription elongation. Using hybridization methods developed to detect microRNAs, Goldman et al. (p. 927) directly detected products of abortive initiation in bacterial cells in vivo. Abortive initiation increased when interactions between RNA polymerase and the promoter were strengthened or when transcription was prevented. Thus, products of abortive initiation may help to regulate gene expression. RNA polymerase engages in abortive transcription in bacteria, a process that may help to regulate gene expression. During transcription initiation in vitro, prokaryotic and eukaryotic RNA polymerase (RNAP) can engage in abortive initiation—the synthesis and release of short (2 to 15 nucleotides) RNA transcripts—before productive initiation. It has not been known whether abortive initiation occurs in vivo. Using hybridization with locked nucleic acid probes, we directly detected abortive transcripts in bacteria. In addition, we show that in vivo abortive initiation shows characteristics of in vitro abortive initiation: Abortive initiation increases upon stabilizing interactions between RNAP and either promoter DNA or sigma factor, and also upon deleting elongation factor GreA. Abortive transcripts may have functional roles in regulating gene expression in vivo.

Interactions between RNA polymerase and the “core recognition element” counteract pausing

Pausing for control of gene expression Pausing during gene transcription can play a critical role in gene regulation. Vvedenskaya et al. mapped pause sites across the whole genome in actively growing Escherichia coli (see the Perspective by Roberts). Thousands of undocumented pause sites were identified across well-transcribed genes, allowing the definition of a consensus pause sequence that is dependent on specific interactions of RNA polymerase with the DNA template and nascent RNA transcript. Science, this issue p. 1285; see also p. 1226 An in vivo transcriptional pause consensus sequence determined in Escherichia coli is functional across prokaryotes. [Also see Perspective by Roberts] 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 RNAP 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 posttranslocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.

Regulation of IcsP, the Outer Membrane Protease of the Shigella Actin Tail Assembly Protein IcsA, by Virulence Plasmid Regulators VirF and VirB

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.

Differential Regulation by Magnesium of the Two MsbB Paralogs of Shigella flexneri

Shigella flexneri, a gram-negative enteric pathogen, is unusual in that it contains two nonredundant paralogous genes that encode the myristoyl transferase MsbB (LpxM) that catalyzes the final step in the synthesis of the lipid A moiety of lipopolysaccharide. MsbB1 is encoded on the chromosome, and MsbB2 is encoded on the large virulence plasmid present in all pathogenic shigellae. We demonstrate that myristoyl transferase activity due to MsbB2 is detected in limited magnesium medium, but not in replete magnesium medium, whereas that due to MsbB1 is detected under both conditions. MsbB2 increases overall hexa-acylation of lipid A under limited magnesium conditions. Regulation of MsbB2 by magnesium occurs at the level of transcription and is dependent on the conserved magnesium-inducible PhoPQ two-component regulatory pathway. Direct hexanucleotide repeats within the promoter upstream of msbB2 were identified as a putative PhoP binding site, and mutations within the repeats led to diminished PhoP-dependent expression of a transcriptional fusion of lacZ to this promoter. Thus, the virulence plasmid-encoded paralog of msbB is induced under limited magnesium in a PhoPQ-dependent manner. PhoPQ regulates the response of many Enterobacteriaceae to environmental signals, which include modifications of lipid A that confer increased resistance of the organism to stressful environments and antimicrobial peptides. The findings reported here are the first example of gene duplication in which one paralog has selectively acquired the mechanism for differential regulation by PhoPQ. Our findings provide molecular insight into the mechanisms by which each of the two MsbB proteins of S. flexneri likely contributes to pathogenesis.

Modulation of an Outer Membrane Protease Contributes to the Virulence Defect of Shigella flexneri Strains Carrying a Mutation in the virK Locus

ABSTRACT The Shigella actin assembly protein IcsA is removed from the bacterial surface by the protease IcsP. We show that decreased intracellular spreading of virK::Tn10 mutants is due in part to significant increases in IcsP and IcsP-mediated cleavage of IcsA and that IcsP expression is a critical determinant of Shigella virulence.

A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5′ RNA-seq

Transcription initiation that involves the use of a 2- to ~4-nt oligoribonucleotide primer, “primer-dependent initiation,” (PDI) has been shown to be widely prevalent at promoters of genes expressed during the stationary phase of growth in Escherichia coli. However, the extent to which PDI impacts E. coli physiology, and the extent to which PDI occurs in other bacteria is not known. Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation. We further demonstrate using high-throughput sequencing of RNA 5′ ends (5′ RNA-seq) that PDI occurs in the pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V. cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the two organisms. In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T−1A+1 or G−1G+1 (where position +1 corresponds to the position of de novo initiation). Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent process that leads to the preferential generation of the linear dinucleotides 5´-UA-3´ and 5´-GG-3´.

The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo

The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ70, can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ70 can bind in trans to TECs that emanate from either a σ70-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ70 to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ70-dependent promoters. DOI: http://dx.doi.org/10.7554/eLife.10514.001