Pathological R-loops in bacteria from engineered expression of endogenous antisense RNAs whose synthesis is ordinarily terminated by Rho.

In many bacteria, the essential factors Rho and NusG mediate termination of synthesis of nascent transcripts (including antisense RNAs) that are not being simultaneously translated. It has been proposed that in Rho's absence toxic RNA-DNA hybrids (R-loops) may be generated from nascent untranslated transcripts, and genome-wide mapping studies in Escherichia coli have identified putative loci of R-loop formation from more than 100 endogenous antisense transcripts that are synthesized only in a Rho-deficient strain. Here we provide evidence that engineered expression in wild-type E.

Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli.

Replication of the circular bacterial chromosome is initiated from a locus oriC with the aid of an essential protein DnaA. One approach to identify factors acting to prevent aberrant oriC-independent replication initiation in Escherichia coli has been that to obtain mutants which survive loss of DnaA. Here, we show that a ΔrecD mutation, associated with attenuation of RecBCD's DNA double strand end-resection activity, provokes abnormal replication and rescues ΔdnaA lethality in two situations: (i) in absence of 5'-3' single-strand DNA exonuclease RecJ, or (ii) when multiple two-ended DNA double strand breaks (DSBs) are generated either by I-SceI endonucleolytic cleavages or by radiomimetic agents phleomycin or bleomycin.

Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E.

RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5'-sensor pocket that renders enzyme activity maximal on 5'-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability.

A new role for Escherichia coli Dam DNA methylase in prevention of aberrant chromosomal replication.

The Dam DNA methylase of Escherichia coli is required for methyl-directed mismatch repair, regulation of chromosomal DNA replication initiation from oriC (which is DnaA-dependent), and regulation of gene expression. Here, we show that Dam suppresses aberrant oriC-independent chromosomal replication (also called constitutive stable DNA replication, or cSDR). Dam deficiency conferred cSDR and, in presence of additional mutations (Δtus, rpoB*35) that facilitate retrograde replication fork progression, rescued the lethality of ΔdnaA mutants.

Genome-wide relationship between R-loop formation and antisense transcription in Escherichia coli.

Transcription termination by Rho is essential for viability in various bacteria, including some major pathogens. Since Rho acts by targeting nascent RNAs that are not simultaneously translated, it also regulates antisense transcription. Here we show that RNase H-deficient mutants of Escherichia coli exhibit heightened sensitivity to the Rho inhibitor bicyclomycin, and that Rho deficiency provokes increased formation of RNA-DNA hybrids (R-loops) which is ameliorated by expression of the phage T4-derived R-loop helicase UvsW.

End of the beginning: elongation and termination features of alternative modes of chromosomal replication initiation in bacteria.

In bacterial cells, bidirectional replication of the circular chromosome is initiated from a single origin (oriC) and terminates in an antipodal terminus region such that movement of the pair of replication forks is largely codirectional with transcription. The terminus region is flanked by discrete Ter sequences that act as polar, or direction-dependent, arrest sites for fork progression. Alternative oriC-independent modes of replication initiation are possible, one of which is constitutive stable DNA replication (cSDR) from transcription-associated RNA-DNA hybrids or R-loops.

Environmental regulation operating at the promoter clearance step of bacterial transcription.

In vivo transcription of the Escherichia coli argO gene, which encodes an arginine (Arg) exporter, requires the LysR-family regulator protein ArgP (previously called IciA) and is induced in the presence of Arg or its naturally occurring antimetabolite analog canavanine. Lysine (Lys) addition, on the other hand, phenocopies an argP mutation to result in the shutoff of argO expression. We now report that the ArgP dimer by itself is able to bind the argO promoter-operator region to form a binary complex, but that the formation of a ternary complex with RNA polymerase is greatly stimulated only in presence of a coeffector.