A simple and unified protocol to purify all seven Escherichia coli RNA polymerase sigma factors.

RNA polymerase sigma factors are indispensable in the process of bacterial transcription. They are responsible for a given gene's promoter region recognition on template DNA and hence determine specificity of RNA polymerase and play a significant role in gene expression regulation. Here, we present a simple and unified protocol for purification of all seven Escherichia coli RNA polymerase sigma factors.

Estimates of Rel, Mesh1, and SAH Hydrolysis of (p)ppGpp and (p)ppApp by Thin Layer Chromatography and NADP/NADH Coupled Assays.

The Mesh1 class of hydrolases found in bacteria, metazoans and humans was discovered as able to cleave an intact pyrophosphate residue esterified on the 3'hydroxyl of (p)ppGpp in a Mn dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from and human MESH1 also extends to the (p)ppApp purine analogs. More importantly, we developed real time enzymatic assays, coupling ppNpp hydrolysis to NADH oxidation and pppNpp hydrolysis to NADP reduction, which facilitate estimation of kinetic constants.

Autoregulation of Expression Relies on GraL Rather than on Promoter Region.

GreA is a well-characterized transcriptional factor that acts primarily by rescuing stalled RNA polymerase complexes, but has also been shown to be the major transcriptional fidelity and proofreading factor, while it inhibits DNA break repair. Regulation of gene expression itself is still not well understood. So far, it has been shown that its expression is driven by two overlapping promoters and that leader encodes a small RNA (GraL) that is acting on mRNA.

[50th anniversary of discovering „magic spots” – the latest advances in (p)ppGpp research].

About 50 years ago, "magic spots" - mediators of the bacterial stringent response, were discovered and were later identified as guanosine tetra- and pentaphosphate (ppGpp and pppGpp, jointly referred to as (p)ppGpp). At first, it seemed that stringent response is associated only with bacterial response to amino acid starvation, however, it soon turned out that (p)ppGpp is synthesized in response to other stresses as well. The mentioned alarmones are found to exist in all known bacterial species, as well as in plants.

Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro.

Precise regulation of gene expression is crucial for bacteria to respond to changing environmental conditions. In addition to protein factors affecting RNA polymerase (RNAP) activity, second messengers play an important role in transcription regulation, such as well-known effectors of the stringent response: guanosine 5'triphosphate-3'diphosphate and guanosine 3', 5'-bis(diphosphate) [(p)ppGpp]. Although much is known about importance of the 5' and 3' moieties of (p)ppGpp, the role of the guanine base remains somewhat cryptic.

A search for the in trans role of GraL, an Escherichia coli small RNA.

Small RNA are very important post-transcriptional regulators in both, bacteria and eukaryotes. One of such sRNA is GraL, encoded in the greA leader region and conserved among enteric bacteria. Here, we conducted a bioinformatics search for GraL's targets in trans and validated our findings in vivo by constructing fusions of probable targets with lacZ and measuring their activity when GraL was overexpressed.