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ABSTRACT: Prokaryotic and eukaryotic RNA polymerases can use 2- to ∼4-nt RNAs, "nanoRNAs," to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.
Genes & development 07/2012; 26(13):1498-507. · 12.08 Impact Factor
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ABSTRACT: 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 oligoribonucleotides 2 to ∼4 nt in length, "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 only NTPs. 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.
Molecular cell 06/2011; 42(6):817-25. · 14.61 Impact Factor
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ABSTRACT: 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.
Science 06/2009; 324(5929):927-8. · 31.20 Impact Factor
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ABSTRACT: 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.
Journal of bacteriology 06/2008; 190(10):3526-37. · 3.94 Impact Factor
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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.
Infection and Immunity 03/2005; 73(2):1217-20. · 4.16 Impact Factor
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ABSTRACT: 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.
Journal of Bacteriology 03/2004; 186(3):699-705. · 3.83 Impact Factor
