DNA recognition by a σ(54) transcriptional activator from Aquifex aeolicus.

Transcription initiation by bacterial σ(54)-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain (DBD). The structurally characterized DBDs from activators all belong to the Fis (factor for inversion stimulation) family of helix-turn-helix DNA-binding proteins.

The TFIIB tip domain couples transcription initiation to events involved in RNA processing.

TFIIB is the only factor within the multimegadalton transcription complex that is obligatorily required to undergo dissociation and re-association with each round of mRNA transcription. Here we show that a six-amino acid human TFIIB tip region is needed for appropriate levels of serine 5 C-terminal domain phosphorylation and mRNA capping and for retention of the required elongation factor TFIIF. We suggest that the broad functions of this tiny region are used to suppress transcription noise by restricting functional RNA synthesis from non-promoter sites on the genome, which will not contain TFIIB.

Osmotic Stress.

Escherichia coli and Salmonella encounter osmotic pressure variations in natural environments that include host tissues, food, soil, and water. Osmotic stress causes water to flow into or out of cells, changing their structure, physics, and chemistry in ways that perturb cell functions. E.

Remodeling and activation of Escherichia coli RNA polymerase by osmolytes.

The ability of bacteria to survive environmental stresses and colonize the gastrointestinal tract depends on adaptation to high osmolarity. The adaptation involves global reprogramming of gene expression, including inhibition of bulk sigma70 RNA polymerase transcription and activation of bulk sigma38 transcription. The activating signal transduction pathways that originate with osmolytes remain to be established.

General stress response signalling: unwrapping transcription complexes by DNA relaxation via the sigma38 C-terminal domain.

Escherichia coli responds to stress by a combination of specific and general transcription signalling pathways. The general pathways typically require the master stress regulator sigma38 (rpoS). Here we show that the signalling from multiple stresses that relax DNA is processed by a non-conserved eight-amino-acid tail of the sigma 38 C-terminal domain.

Control of the timing of promoter escape and RNA catalysis by the transcription factor IIb fingertip.

Transcription factor IIB (TFIIB) recruits RNA polymerase II to promoters and inserts a finger domain into its active site, with unknown consequences. Here we show that that the tip of this finger is important for two transcription initiation functions. First, TFIIB acts as a catalytic cofactor for initial RNA bond formation.

Regulation of transcription by acetate in Escherichia coli: in vivo and in vitro comparisons.

Acetate, even at neutral pH, induces changes in gene expression that allow Escherichia coli to adapt to the diverse chemical stresses of the gastrointestinal tract. These include differential effects on transcription, including both activation and repression. The in vivo studies presented here show that cyclopropane fatty acid synthase transcription induced by neutral acetate proceeds via both the sigma70 and sigma38 forms of RNA polymerase.

Poising of Escherichia coli RNA polymerase and its release from the sigma 38 C-terminal tail for osmY transcription.

Bacteria must adapt their transcription to overcome the osmotic stress associated with the gastrointestinal tract of their host. This requires the sigma 38 (rpoS) form of RNA polymerase. Here, chromatin immunoprecipitation experiments show that activation is associated with a poise-and-release mechanism in vivo.

Escherichia coli pfs transcription: regulation and proposed roles in autoinducer-2 synthesis and purine excretion.

Pfs expression is required for several metabolic pathways and limits the production of autoinducer-2, a molecule proposed to play a central role in interspecies quorum sensing. The present study reveals physiological conditions and promoter DNA elements that regulate Escherichia coli pfs transcription. Pfs transcription is shown to rely on both sigma 70 and sigma 38 (rpoS), and the latter is subject to induction that increases pfs expression.

Potassium glutamate as a transcriptional inhibitor during bacterial osmoregulation.

Potassium glutamate accumulates upon hyper-osmotic shock and serves as a temporary osmoprotectant. This salt leads to transcriptional activation of sets of genes that allow the cell to achieve long-term adaptation to high osmolarity. The current experiments show that potassium glutamate also acts as an inhibitor of bulk cellular transcription.

Osmolyte-induced transcription: -35 region elements and recognition by sigma38 (rpoS).

In order to meet osmotic challenges in the gastrointestinal tract, enteric bacteria rapidly accumulate salts of glutamate and other weak organic acids. The ensuing transcriptional activation is mediated by unknown elements at sigma38 (rpoS)-dependent promoters. Here we identify DNA elements needed for high levels of transcription in the presence of salt and acetate and show that they are associated with the -35 regions of target promoters.

TFIIH XPB mutants suggest a unified bacterial-like mechanism for promoter opening but not escape.

DNA helicases open the duplex during DNA replication, repair and transcription. However, RNA polymerase II is the only member of its family with this requirement; RNA polymerases I and III and bacterial RNA polymerases open DNA without a helicase. In this report, characterization of XPB mutants indicates that its helicase activity is not used for RNA polymerase II promoter opening, which is instead driven by its ATPase activity.

Stimulation of the XPB ATP-dependent helicase by the beta subunit of TFIIE.

TFIIE and TFIIH are essential for the promoter opening and escape that occurs as RNA polymerase II transits into early elongation. XPB, a subunit of TFIIH, contains an ATP-dependent helicase activity that is used in both of these processes. Here, we show that the smaller beta subunit of TFIIE stimulates the XPB helicase and ATPase activities.

Escherichia coli ribosomal RNA transcription: regulatory roles for ppGpp, NTPs, architectural proteins and a polymerase-binding protein.

Ribosomal RNA transcription can limit the rate of Escherichia coli growth and is subject to complex regulation. Somehow, the cell is able to sense the general nutritional environment and adjust rRNA transcription so that an appropriate number of ribosomes is produced. This review discusses the current state of affairs, including recent information about the involvement of two nucleotide regulators, two architectural protein regulators, one new co-regulator and stalled ribosomes.

Nucleotide-dependent isomerization of Escherichia coli RNA polymerase.

During promoter engagement, RNA polymerase must change conformation or isomerize to its active form. These data show that high concentrations of nucleotides assist this isomerization. When binding to fork junction DNA probes is monitored, isomerization can occur without the need for the DNA that overlaps the transcription start site.

The Schizosaccharomyces pombe open promoter bubble: mammalian-like arrangement and properties.

The fission yeast Schizosaccharomyces pombe is often used as a genetic system to model processes that apply to higher cells. Here S.pombe was used to study promoter DNA opening and transcription initiation by RNA polymerase II.

Osmo-regulation of bacterial transcription via poised RNA polymerase.

Adaptation to high-salt environments is critical for the survival of a wide range of cells, especially for pathogenic bacteria that colonize the animal gut and urinary tract. The adaptation strategy involves production of the salt potassium glutamate, which induces a specific gene expression program that produces electro-neutral osmolytes while inhibiting general sigma(70) transcription. These data show that in Escherichia coli potassium glutamate stimulates transcription by disengaging inhibitory polymerase interactions at a sigma(38) promoter.

Mechanism of stimulation of ribosomal promoters by binding of the +1 and +2 nucleotides.

The rate of transcription of Escherichia coli ribosomal RNA promoters is central to adjusting the cellular growth rate to nutritional conditions. The +1 initiating nucleotide and ppGpp are regulatory effectors of these promoters. The data herein show that in vitro transcription is also regulated by the +2 nucleotide.

Roles for inhibitory interactions in the use of the -10 promoter element by sigma 70 holoenzyme.

A panel of seven -10 region DNA mutants was tested for holoenzyme binding against a panel of 13 region 2 mutants of sigma 70. No patterns were noticed that would indicate unique interactions between individual amino acids and individual -10 region bases. Instead, certain amino acid substitutions led to increased holoenzyme binding to DNA, implying that the wild type interactions are associated with an inhibitory component.

Open complex formation in vitro by sigma38 (rpoS) RNA polymerase: roles for region 2 amino acids.

Non-functional mutants of sigma(38)(sigma(S)) were studied in vitro to identify the nature of their defects. Mutations in four amino acids led to severe defects in DNA binding and enzyme isomerization with promoter fork junction probes containing single-stranded non-template DNA. The same properties were previously seen with DNA mutations at the fork junction, implying that sigma:DNA interactions at the fork junction are used both for DNA binding and enzyme isomerization.

Effect of DNA bases and backbone on sigma70 holoenzyme binding and isomerization using fork junction probes.

Abasic substitutions in the non-template strand and promoter sequence changes were made to assess the roles of various promoter features in sigma70 holoenzyme interactions with fork junction probes. Removal of -10 element non-template single strand bases, leaving the phosphodiester backbone intact, did not interfere with binding. In contrast these abasic probes were deficient in promoting holoenzyme isomerization to the heparin resistant conformation.

Promoter use by sigma 38 (rpoS) RNA polymerase. Amino acid clusters for DNA binding and isomerization.

Sigma(38) is a non-essential but highly homologous member of the sigma(70) family of transcription factors. In vitro mutagenesis and in vivo screening were used to identify 22 critical amino acids in the promoter interaction domain of Escherichia coli sigma(38). Electrophoretic mobility shift assay studies showed that residues involved in duplex DNA binding largely segregated into distinct regions that coincided with those of sigma(70).

New roles for conserved regions within a sigma 54-dependent enhancer-binding protein.

23 amino acid substitutions were made in the C7 and C3 regions of pspFDeltaHTH, a protein required to convert sigma(54) closed promoter complexes to open complexes. These mutants were assayed for transcriptional competence, for the ability to hydrolyze ATP, for their multimerization state, and for their ability to interact with sigma(54) and its holoenzyme. C7 region mutants caused the protein to assume a compact form.

In vitro transcription and start site selection in Schizosaccharomyces pombe.

We have used the fission yeast Schizosaccharomyces pombe to establish both a biochemical and genetic system to study the roles of general transcription factors in transcription initiation. Extracts were prepared that faithfully transcribed S. pombe promoters and the results confirm that, in contrast to the budding yeast Saccharomyces cerevisiae, in vitro transcription in S.