In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways.

Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3' fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators.

Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA.

MicF is a textbook example of a small regulatory RNA (sRNA) that acts on a trans-encoded target mRNA through imperfect base pairing. Discovery of MicF as a post-transcriptional repressor of the major Escherichia coli porin OmpF established the paradigm for a meanwhile common mechanism of translational inhibition, through antisense sequestration of a ribosome binding site. However, whether MicF regulates additional genes has remained unknown for almost three decades. Here, we have harnessed the new superfolder variant of GFP for reporter-gene fusions to validate newly predicted targets of MicF in Salmonella. We show that the conserved 5' end of MicF acts by seed pairing to repress the mRNAs of global transcriptional regulator Lrp, and periplasmic protein YahO, while a second targeting region is also required to regulate the mRNA of the lipid A-modifying enzyme LpxR. Interestingly, MicF targets lpxR at both the ribosome binding site and deep within the coding sequence. MicF binding in the coding sequence of lpxR decreases mRNA stability through exacerbating the use of a native RNase E site proximal to the short MicF-lpxR duplex. Altogether, this study assigns the classic MicF sRNA to the growing class of Hfq-associated regulators that use diverse mechanisms to impact multiple loci.

Bacterial DNA topology and infectious disease.

The gram-negative bacterium Escherichia coli and its close relative Salmonella enterica have made important contributions historically to our understanding of how bacteria control DNA supercoiling and of how supercoiling influences gene expression and vice versa. Now they are contributing again by providing examples where changes in DNA supercoiling affect the expression of virulence traits that are important for infectious disease. Available examples encompass both the earliest stages of pathogen-host interactions and the more intimate relationships in which the bacteria invade and proliferate within host cells. A key insight concerns the link between the physiological state of the bacterium and the activity of DNA gyrase, with downstream effects on the expression of genes with promoters that sense changes in DNA supercoiling. Thus the expression of virulence traits by a pathogen can be interpreted partly as a response to its own changing physiology. Knowledge of the molecular connections between physiology, DNA topology and gene expression offers new opportunities to fight infection.

H-NS silences gfp, the green fluorescent protein gene: gfpTCD is a genetically Remastered gfp gene with reduced susceptibility to H-NS-mediated transcription silencing and with enhanced translation.

The bacterial nucleoid-associated protein H-NS, which preferentially targets and silences A+T-rich genes, binds the ubiquitous reporter gene gfp and dramatically reduces local transcription. We have redesigned gfp to reduce H-NS-mediated transcription silencing and simultaneously improve translation in vivo without altering the amino acid sequence of the GFP protein.

DNA relaxation-dependent phase biasing of the fim genetic switch in Escherichia coli depends on the interplay of H-NS, IHF and LRP.

Reversible inversion of the DNA element fimS is responsible for the phase variable expression of type 1 fimbriae in Escherichia coli. The FimB tyrosine integrase site-specific recombinase inverts fimS in the on-to-off and off-to-on directions with approximately equal efficiencies. However, when DNA supercoiling is relaxed, fimS adopts predominantly the on orientation. This orientational bias is known to require binding of the nucleoid-associated protein LRP within fimS. Here we show that binding of the IHF protein to a site immediately adjacent to fimS is also required for phase-on orientational bias. In the absence of both LRP and IHF binding, fimS adopts the off orientation and the H-NS protein is required to maintain this alternative orientational bias. Thus, fimS has three Recombination Directionality Factors, H-NS, IHF and LRP. The relevant H-NS binding site straddles the left inverted repeat in phase-off fimS and this site is disrupted when fimS inverts to the on orientation. The inversion of fimS with the associated creation and removal of an H-NS binding site required for DNA inversion biasing represents a novel mechanism for modulating the interaction of H-NS with a DNA target and for influencing a site-specific recombination reaction.

Use of aptamer tagging to identify in vivo protein binding partners of small regulatory RNAs.

Small regulatory RNAs (sRNAs) are short, generally noncoding RNAs that act posttranscriptionally to control target gene expression. Over the past 10 years there has been a rapid expansion in the discovery and characterization of sRNAs in a diverse range of bacteria. Paradigm shifts in our understanding of the breadth of posttranscriptional control by sRNAs were achieved in a number of pioneering studies that involved immunoprecipitation of a known RNA chaperone, the near-ubiquitous Hfq, followed by sequencing to identify novel putative regulators and targets. To perform the converse experiment, we previously developed a method which uses an aptamer-tagged sRNA to allow purification of in vivo assembled RNA-protein complexes and subsequent identification of bound proteins. We successfully implemented this protocol using the Hfq-associated sRNA, InvR, tagged with a tandem repeat of the commonly used MS2-aptamer. Incorporation of the aptamer had no effect on sRNA stability or activity. InvR-MS2 could be effectively purified along with associated proteins, such as Hfq, using maltose binding protein fused to the MS2 coat protein (MBP-MS2) immobilized on an amylose column. Mass-spectroscopy was also used to identify previously uncharacterized protein partners. These results have been described previously (Said et al., Nucleic Acids Res 37:e133, 2009) and thus the figures presented here are intended solely as an illustrative guide to complement this detailed step-by-step protocol.