SummaryThe Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have sequence similarity and broadly similar sequence specificity. Whereas the absence of Rne normally is lethal, we show here that E. coli bacteria that lack the rne gene can be made viable by overexpression of Rng. Rng-complemented cells accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase P (i.e. M1 RNA), indicating that normal processing of these Rnecleaved RNAs was not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated from precursors by RNase G cleavage in vitro. Using DNA microarrays containing 4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose steady-state level was affected by Rne, Rng or the N-terminal catalytic domain of RNase E. Most transcript species affected by RNase E deficiency were also elevated in an rne deletion mutant complemented by Rng. However, approximately 100 mRNAs that accumulated in Rne-deficient cells were decreased by rng-complemention, thus identifying targets whose processing or degradation may be the basis for RNase E essentiality. Remarkably prominent in this group were mRNAs implicated in energygenerating pathways or in the synthesis or degradation of macromolecules.
SummaryPrevious work has detected an RNase E-like endoribonucleolytic activity in cell extracts obtained from Streptomyces . Here, we identify a Streptomyces coelicolor gene, rns , encoding a 140 kDa protein (RNase ES) that shows endoribonucleolytic cleavage specificity characteristic of RNase E, confers viability on and allows propagation of Escherichia coli cells lacking RNase E and accomplishes RNase E-like regulation of plasmid copy number in E. coli . However, notwithstanding its complementation of rne -deleted E. coli , RNase ES did not accurately process 9S rRNA from E. coli . Additionally, whereas RNase E is normally required for E. coli survival, rns is not an essential gene in S. coelicolor . Deletion analysis mapped the catalytic domain of RNase ES near its centre and showed that regions located near the RNase ES termini interact with an S. coelicolor homologue of polynucleotide phosphorylase (PNPase) -a major component of E. coli RNase E-based degradosomes. The interacting arginine-and proline-rich segments resemble the C-terminally located degradosome scaffold region of E. coli RNase E. Our results indicate that RNase ES is a structurally shuffled RNase E homologue showing evolutionary conservation of functional RNase E-like enzymatic activity, and suggest the existence of degradosome-like complexes in Gram-positive bacteria.
SummaryIn Escherichia coli the initial step in the processing or decay of many messenger and structural RNAs is mediated by the endonuclease RNase E, which forms the core of a large RNA-catalysis machine termed the degradosome. Previous experiments have identified a protein that globally modulates RNA abundance by binding to RNase E and regulating its endonucleolytic activity. Here we report the discovery of RraB, which interacts with a different site on RNase E and interferes with cleavage of a different set of transcripts. We show that expression of RraA or RraB in vivo is accompanied by dramatic, distinct, and inhibitorspecific changes in degradosome composition -and that these are in turn associated with alterations in RNA decay and global transcript abundance profiles that are dissimilar to the profile observed during simple RNase E deficiency. Our results reveal the existence of endonuclease binding proteins that modulate the remodelling of degradosome composition in bacteria and argue that such degradosome remodelling is a mechanism for the differential regulation of RNA cleavages in E. coli.
Macrolide-specific efflux pump MacAB-TolC has been identified in diverse Gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC ␣-barrel to investigate the role of the TolC ␣-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA ␣-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the ␣-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.Drug resistance of microbial pathogens presents an increasing threat to public health (1). In Gram-negative pathogens, high levels of intrinsic or acquired drug resistance are conferred by three-component multidrug efflux pumps, which are composed of the inner membrane transporter, the outer membrane factor (OMF), and the periplasmic membrane fusion protein (MFP) 4 (2-5). These tripartite complexes span the entire twomembrane envelope of Gram-negative bacteria and expel various molecules into the medium, utilizing a proton gradient or ATP hydrolysis. The inner membrane transporters belong to one of three structurally dissimilar superfamilies of proteins: resistance-nodulation-cell division (RND), ATP-binding cassette (ABC), or major facilitator. The inner membrane transporters expel the substrates through the central channel of the OMF, as exemplified by Escherichia coli TolC, which spans the outer membrane (6). The MFP, which connects the other two components in the periplasm, is also essential for the function of the efflux pump.In E. coli, AcrAB-TolC acts as a major multidrug efflux pump (7-9), where AcrB is the RND-type inner membrane transporter and AcrA belongs to MFP. The homotrimeric TolC is embedded in the outer membrane and continues ϳ100 Å into the periplasmic space as an ␣-barrel composed of six ␣-hairpins that form the wall of a 35-Å inner-diameter cylindrical channel (10). The TolC channel is closed at the aperture end and the channel opening is induced only in the presence of the other components, the mechanism of which remains to be determined at the molecular level.The MacAB-TolC pump has been identified in E. coli; the inner membrane transporter MacB belongs to non-canonic ABC-type transporters (8,9,11,12), and MFP MacA shares structural similarity with AcrA (sequence similarity 44%) (13). Overproduction of MacAB results in increased resistance to the macrolide antibiotics in macrolide-susceptible AcrAB-deficient E. coli (8, 9, 11).The s...
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