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Transcriptional regulation for genome expression determines growth and adaptation of single-cell bacteria that are directly exposed to environment. The transcriptional apparatus in Escherichia coli K-12 is composed of RNA polymerase core enzyme and two groups of its regulatory proteins, seven species of promoter-recognition subunit sigma and about 300 species of transcription factors. The identification of regulatory targets for all these regulatory proteins is critical toward understanding the genome regulation as a whole. For this purpose, we performed a systematic search in vitro of the whole set of binding sites for each factor by gSELEX system. This review summarizes the accumulated knowledge of regulatory targets for more than 150 TFs from E. coli K-12. Overall TFs could be classified into four families: nucleoid-associated bifunctional TFs; global regulators; local regulators; and single-target regulators, in which the regulatory functions remain uncharacterized for the nucleoid-associated TFs. Here we overview the regulatory targets of two nucleoid-associated TFs, H-NS and its paralog StpA, both together playing the silencing role of a set of non-essential genes. Participation of LeuO and other global regulators have been indicated for the anti-silencing. Finally, we propose the hierarchy of TF network as a key framework of the bacterial genome regulation. [ABSTRACT FROM AUTHOR]

Transcription and translation in growing phase of Escherichia coli , the best-studied model prokaryote, are coupled and regulated in coordinate fashion. Accordingly, the growth rate-dependent control of the synthesis of RNA polymerase (RNAP) core enzyme (the core component of transcription apparatus) and ribosomes (the core component of translation machinery) is tightly coordinated to keep the relative level of transcription apparatus and translation machinery constant for effective and efficient utilization of resources and energy. Upon entry into the stationary phase, transcription apparatus is modulated by replacing RNAP core-associated sigma (promoter recognition subunit) from growth-related RpoD to stationary-phase-specific RpoS. The anti-sigma factor Rsd participates for the efficient replacement of sigma, and the unused RpoD is stored silent as Rsd–RpoD complex. On the other hand, functional 70S ribosome is transformed into inactive 100S dimer by two regulators, ribosome modulation factor (RMF) and hibernation promoting factor (HPF). In this review article, we overview how we found these factors and what we know about the molecular mechanisms for silencing transcription apparatus and translation machinery by these factors. In addition, we provide our recent findings of promoter-specific transcription factor (PS-TF) screening of the transcription factors involved in regulation of the rsd and rmf genes. Results altogether indicate the coordinated regulation of Rsd and RMF for simultaneous hibernation of transcription apparatus and translation machinery. [ABSTRACT FROM AUTHOR]

The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by the sigma subunit. The model prokaryote Escherichia coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. For identification of the “constitutive promoters” that are recognized by each RNAP holoenzyme alone in the absence of other supporting factors, we have performed the genomic SELEX screening in vitro for their binding sites along the E. coli K-12 W3110 genome using each of the reconstituted RNAP holoenzymes and a collection of genome DNA segments of E. coli K-12. The whole set of constitutive promoters for each RNAP holoenzyme was then estimated based on the location of RNAP-binding sites. The first successful screening of the constitutive promoters was achieved for RpoD (σ70), the principal sigma for transcription of growth-related genes. As an extension, we performed in this study the screening of constitutive promoters for four minor sigma subunits, stationary-phase specific RpoS (σ38), heat-shock specific RpoH (σ32), flagellar-chemotaxis specific RpoF (σ28) and extra-cytoplasmic stress-response RpoE (σ24). The total number of constitutive promoters were: 129~179 for RpoS; 101~142 for RpoH; 34~41 for RpoF; and 77~106 for RpoE. The list of constitutive promoters were compared with that of known promoters identified in vivo under various conditions and using varieties of E. coli strains, altogether allowing the estimation of “inducible promoters” in the presence of additional supporting factors. [ABSTRACT FROM AUTHOR]

Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3’ proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed independent of rRNA synthesis under specific conditions where further synthesis of ribosomes is not needed. [ABSTRACT FROM AUTHOR]

The promoter selectivity of Escherichia coli RNA polymerase is determined by the sigma subunit with promoter recognition activity. The model prokaryote Escherichia coli contains seven species of the sigma subunit, each recognizing a specific set of promoters. The major sigma subunit, sigma-70 encoded by rpoD, plays a major role in transcription of growth-related genes. Concomitant with the increase in detection of promoters functioning in vivo under various stressful conditions, the variation is expanding in the consensus sequence of RpoD promoters. In order to identify the canonical sequence of “constitutive promoters” that are recognized by the RNA polymerase holoenzyme containing RpoD sigma in the absence of supporting transcription factors, an in vitro mixed transcription assay was carried out using a whole set of variant promoters, each harboring one base replacement, within the model promoter with the conserved -35 and -10 sequences of RpoD promoters. The consensus sequences, TTGACA(-35) and TATAAT(-10), were identified to be ideal for the maximum level of open complex formation and the highest rate of promoter opening, respectively. For identification of the full range of constitutive promoters on the E. coli genome, a total of 2,701 RpoD holoenzyme-binding sites were identified by Genomic SELEX screening, and using the reconfirmed consensus promoter sequence, a total of maximum 669 constitutive promoters were identified, implying that the majority of hitherto identified promoters represents the TF-dependent “inducible promoters”. One unique feature of the constitutive promoters is the high level of promoter sequence conservation, about 85% carrying five-out-of-six agreements with -35 or -10 consensus sequence. The list of constitutive promoters provides the community resource toward estimation of the inducible promoters that operate under various stressful conditions in nature. [ABSTRACT FROM AUTHOR]

Influenza virus RNA polymerase is composed of three virus-coded proteins, and is involved in both transcription and replication of the negative-strand genome RNA. Subunit PB1 plays key roles in both the RNA polymerase assembly and the catalytic function of RNA polymerization. Using yeast two-hybrid screening, a HeLa cell protein with the molecular mass of 45 kDa was identified. After cloning and sequencing, this protein was identified to be Ebp1, ErbB3-binding protein. Epb1 specifically interacts with PB1 both in vitro and in vivo, and Epb1 contact site on PB1 was mapped at its binding site of transcription primers. Ebp1 was found to interfere with in vitro RNA synthesis by influenza virus RNA polymerase (3P complex), but no inhibition was observed for capped RNA endonuclease and RNA-cap binding, the intrinsic activities of RNA polymerase. Since inhibition was not observed against other nucleic acid polymerases tested, we propose that Ebp1 is a selective inhibitor of influenza viral RNA polymerase. Accordingly over-expression of Ebp1 interfered with virus production. The PB1-contact site on Ebp1 overlaps with the interaction site with ErbB3 (epidermal receptor tyrosine kinase), androgen receptor (AR) and retinoblastoma gene product (Rb), which are involved in controlling cell proliferation and differentiation. [ABSTRACT FROM AUTHOR]

The rpoZ gene for the omega subunit of Escherichia coli RNA polymerase constitutes single operon with the spoT gene, which is responsible for the maintenance of stringent response under nutrient starvation conditions. To identify the physiological role of the omega subunit, we compared the gene expression profile of wild-type Escherichia coli with that of an rpoZ deleted strain by microarray analysis using an E. coli DNA chip. Here we report on a set of genes which show changes in expression profile following the removal of rpoZ. We have seen that relA, which is responsible for the synthesis of the stringent factor ppGpp and many ribosomal proteins, exhibited noticeable changes in mRNA levels and were therefore further analyzed for their expression using a GFP/RFP two-fluorescent protein promoter assay vector. In the absence of rpoZ, the promoter for the relA gene was severely impaired, but the promoters from the ribosomal protein genes were not affected as much. Taking these results together we propose that the omega subunit is involved in regulation of the relA gene, but induction of the stringently controlled genes in the absence of rpoZ is, at least in part, attributable to a decrease in ppGpp level. [ABSTRACT FROM AUTHOR]

The general transcription factor TFIIE plays essential roles in transcription by RNA polymerase II (PolII). Despite recent progress, the elucidation of its precise mechanisms including biological functions awaits further characterization. We report the isolation and characterization ofSchizosaccharomyces pombeTFIIE (spTFIIE). Like human and other eukaryotic TFIIE proteins, spTFIIE consists ofα andβ subunits and the genes encoding both subunits are essential for viability. Chromatin immunoprecipitation assays demonstrated that spTFIIE localizes to promotersin vivo. Mutational analysis of the C-terminal basic helix-loop region of TFIIEβ, which is involved in the transition from transcription initiation to elongation, revealed that transcription-defective mutants affected in this region are also cold sensitive. The spTFIIEβ subunit binds both spTFIIEβ and spTFIIEα but spTFIIEα binds only spTFIIEβ. These results indicate that TFIIE forms anα2β2 heterotetramer in which twoαβ heterodimers are connected viaβ subunits. Further analysis of binding specificities showed that spTFIIEβ binds the Rpb2 and Rpb12 subunits of PolII, whereas spTFIIEα predominantly binds Rpb5, which is located at the clamp region and changes conformation upon transcription initiation. [ABSTRACT FROM AUTHOR]

Abstract: RNA polymerase II (pol II) purified from the fission yeast Schizosaccharomyces pombe was previously reported to be associated with the general transcription factor TFIIF and the C-terminal domain phosphatase Fcp1, as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has recently been implicated in transcriptional activation in human cells. Here, we provide evidence that the Rpb7 subunit of pol II interacts with GAPDH. Two-hybrid screen identified GAPDH as an Rpb7-binding protein. In addition, GAPDH was affinity-purified from S. pombe extract by using an Rpb4/Rpb7-coupled column. We also identified actin as a pol II-associated protein and revealed the interaction between actin and Rpb7. [Copyright &y& Elsevier]

There are few existing indications that strain variation in prokaryotic gene regulation is common or has evolutionary advantage. In this study, we report on isolates of Escherichia coil with distinct ratios of sigma factors (RpoD, σD, or sigma;70 and RpoS or σS) that affect transcription initiated by RNA polymerase. Both laboratory E. coli K-12 lineages and nondomesticated isolates exhibit strain-specific endogenous levels of RpoS protein. We demonstrate that variation in genome usage underpins intraspecific variability in transcription patterns, resistance to external stresses, and the choice of beneficial mutations under nutrient limitation. Most unexpectedly, RpoS also controlled strain variation with respect to the metabolic capability of bacteria with more than a dozen carbon sources. Strains with higher σS levels were more resistant to external stress but metabolized fewer substrates and poorly competed for low concentrations of nutrients. On the other hand, strains with lower σS levels had broader nutritional capabilities and better competitive ability with low nutrient concentrations but low resistance to external stress. In other words, RpoS influenced both r and K strategist functions of bacteria simultaneously. The evolutionary principle driving strain variation is proposed to be a conceptually novel trade-off that we term SPANC (for "self-preservation and nutritional competence"). The availability of multiple SPANC settings potentially broadens the niche occupied by a species consisting of individuals with narrow specialization and reveals an evolutionary advantage offered by polymorphic regulation. Regulatory diversity is likely to be a significant contributor to complexity in a bacterial world in which multiple sigma factors are a universal feature. [ABSTRACT FROM AUTHOR]

Influenza virus RNA polymerase is composed of three viral P proteins (PB1, PB2 and PA) and involved in both transcription and replication of the viral RNA genome. Using recombinant baculoviruses, we constructed the PA–PB1–PB2 (3P) complex and two kinds of 2P complex (PA–PB1 and PB1–PB2). The 3P complex is not fully active but vRNA acts as “RNA effector” for conversion of it into an active form. The cap structure (7mGpppAm) also plays an allosteric effector for RNA polymerase activation. The PB1–PB2 complex carries the catalytic specificity of transcriptase, while the PA–PB1 complex harbours the replicase specificity. We propose that the 3P complex behaves as the PB1–PB2 complex in transcription and the PA–PB1 complex in replication. A host factor(s) seems to be involved in the functional conversion of 3P complex. The candidate host factors have been isolated using yeast two-hybrid screening. [Copyright &y& Elsevier]

Eukaryotic RNA polymerase II (Pol II) transcription apparatus is a multi-protein complex consisting of the RNA polymerase II core enzyme (12 subunits), general transcription factors, the mediator, and some other specific accessory factors with regulatory functions. After genome sequencing was completed, the fission yeast Schizosaccharomyces pombe was recognized as a good model organism to study the Pol II transcription apparatus, because most genetic methods developed with the budding yeast Saccharomyces cerevisiae are applicable but the genetic systems of Sch. pombe, including transcription, are closer to those in higher eukaryotes. Recent studies on components of the Sch. pombe basal transcription machinery not only revealed a number of properties common in other eukaryotes but also illuminated some features unique to Sch. pombe. Convergence of information from both yeasts will provide us with a more general understanding of eukaryotic transcription. [ABSTRACT FROM AUTHOR]

Abstract Background: The RNA polymerase II of the fission yeast Schizosaccharomyces pombe consists of 12 Rpb subunits, of which four (Rpb1, Rpb2, Rpb3 and Rpb11) form the assembly and catalytic core and five (Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12) are shared among RNA polymerases I, II and III. The intracellular levels of three RNA polymerase forms should be interrelated, but the control of RNA polymerase formation remains mostly unknown. Results: To reveal the physiological role and the synthesis control of each Rpb subunit, the intracellular levels of the Rpb proteins were examined in S. pombe growing at various phases under various conditions. Results indicate that the intracellular concentrations of the Rpb proteins stay constant at levels characteristic of the rate and phase of cell growth, and the relative level between the 12 subunits also remains constant, together implying that the intracellular concentration of RNA polymerase II stays constant, as in the case of prokaryotes. As an attempt to gain insights into the activity control of RNA polymerase II, we also analysed the phosphorylation level of the carboxyl-terminal domain (CTD) of the largest subunit Rpb1. Phosphorylated forms of Tyr1 and Thr4 within 29 repeats of the YSPTSPS heptapeptide were detected in both slow-migrating IIo and fast-migrating IIa forms of Rpb1 on SDS-PAGE (polyacrylamide gel electrophoresis). However, phosphorylated Ser2 and Ser5 were identified only in the IIo form, indicating that Ser phosphorylation contributes to the conformational change in CTD. The phosphorylation levels of Ser, Thr and Tyr all vary depending on the cell culture conditions. Conclusion: The intracellular level of RNA polymerase II stays constant, but the amount engaged in transcription cycle varies depending on the culture conditions, as estimated from the sites and levels of phosphorylation of Rpb1 CTD. [ABSTRACT FROM AUTHOR]

Abstract Background: The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure α2 ββ′) associated with one of the σ subunits, required for promoter recognition. Different σ factors compete for core binding. Among the seven σ factors present in E. coli , σ70 controls gene transcription during the exponential phase, whereas σS regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled σS and σ70 , we compared the affinities of both σ factors for core binding and investigated the structural changes in the different subunits involved in the formation of the holoenzymes. Results: Using native polyacrylamide gel electrophoresis, we demonstrate that σS binds to the core enzyme with fivefold reduced affinity compared to σ70 . Using iron chelate protein footprinting, we show that the core enzyme significantly reduces polypeptide backbone solvent accessibility in regions 1.1, 2.5, 3.1 and 3.2 of σS , while increasing the accessibility in region 4.1 of σS . We have also analysed the positioning of σS on the holoenzyme by the proximity-dependent protein cleavage method using σS derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the β and β′ subunits, but not α. Regions 2.5, 3.1 and 3.2 of σS are close to both β and β′ subunits, in agreement with iron chelate protein footprinting data. Conclusions: A comparison between these results using σS and previous data from σ70 indicates similar contact patterns on the core subunits and similar characteristic changes associated with... [ABSTRACT FROM AUTHOR]

In the deoP2 promoter of Escherichia coli, a transcription activator, cAMP-CRP, binds at two sites, centered at -41.5 and -93.5 from the start site of transcription, while a repressor, CytR, binds to a space between the two cAMP-CRP complexes. The mechanisms for the cAMP-CRP-mediated transcription activation and CytR-mediated transcription repression were investigated in vitro using purified components. We classified the deoP2 promoter as a class II cAMP-CRP-dependent promoter, primarily by the action of cAMP-CRP at the downstream site. Interestingly, we also found that deoP2 carries an `UP-element' immediately upstream of the downstream cAMP-CRP site. The UP-element overlaps with the DNA site for CytR. However, it was observed that CytR functions with the RNA polymerase devoid of the C-terminal domain of the a-subunit as well as with intact RNA polymerase. The mechanism of repression by CytR proposed in this study is that the cAMP-CRP bound at -41.5 undergoes an allosteric change upon direct interaction with CytR such that it no longer maintains a productive interaction with the N-terminal domain of α, but instead acts as a repressor to interfere with RNA polymerase acting on deoP2. [ABSTRACT FROM AUTHOR]

The ω subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (α2ββ′). The presence of ω is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an ω-less strain of E. coli, GroEL appears to substitute for ω in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of ω latch on to β′ at its N-terminus and C-terminus. We show here that ω binds only the intact β′ subunit and not the β′ N-terminal domain or β′ C-terminal domain, implying that ω binding requires both these regions of β′. We further show that ω can prevent the aggregation of β′ during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of ω is sufficient for binding and renaturation of β′. CD and functional assays show that this N-terminal fragment retains the structure of native ω and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps α + α → α2 + β → α2β + β′ → α2ββ′. It is shown here that ω participates during the last stage of enzyme assembly when β′ associates with the α2β subassembly. [ABSTRACT FROM AUTHOR]

The RNA polymerase II (Pol II) of Schizosaccharomyces pombe is composed of 12 subunits. Subunit Rpb3 has sequence homology with the N-terminal domain of the prokaryotic α subunit, which plays a key role in RNA polymerase assembly. Together with the Rpb2 (the β homologue) and Rpb11 (the second α homologue) subunits, Rpb3 constitutes a core subassembly (Rpb2–Rpb3–Rpb11) which corresponds to the the α2β assembly intermediate of prokaryotic RNA polymerase. For the functional mapping of Rpb3, we made a collection of 12 heat-sensitive (Ts) or cold-sensitive (Cs) S. pombe mutants, each carrying a single mutation in one of the four conserved regions of Rpb3. The altered functions of six representative Pol II mutants containing the mutant Rpb3 were analyzed in vitro using an improved version of the GAL4-VP16 activator-dependent transcription system catalyzed by S. pombe cell extracts. The transcription activity by the extracts from Rpb3 mutants decreased to varying extents after heat treatment; but the extracts from Rpb3 mutants which had mutations in the eukaryote-specific conserved regions B and C regained their activity by the addition of GAL4-VP16, to a larger extent than those from the region A and D mutants. We propose that both terminal regions (A and D) play important roles in RNA polymerase assembly, while the central portion (regions B and C) is involved in activated transcription. [ABSTRACT FROM AUTHOR]

Investigates the intracellular contents of RNA polymerase II (Pol II) in the fission yeast Schizosaccharomyces (S.) pombe. Quantification of Pol II subunits in cell lysates; Identification of the glycerol gradient centrifugation of S. pombe; Formation of Escherichia coli RNA polymerase.

Discusses the functional modulation of Escherichia coli ribonucleic acid (RNA) polymerase. Replacement of the sigma subunit; Factors affecting selective utilization of transcription apparatus; Activity control of the sigma subunits by anti-sigma factors; Multiple pathways for stationary phase adaptation.

Two distinct classes of RNA polymerase sigma factors (σ) exist in bacteria and are largely unrelated in primary amino acid sequence and their modes of transcription activation. Using tethered iron chelate (Fe-BABE) derivatives of the enhancer-dependent σ54, we mapped several sites of proximity to the β and β′ subunits of the core RNA polymerase. Remarkably, most sites localized to those previously identified as close to the enhancer-independent σ70 and σ38. This indicates a common use of sets of sequences in core for interacting with the two σ classes. Some sites chosen in σ54 for modification with Fe-BABE were positions, which when mutated, deregulate the σ54 holoenzyme and allow activator-independent initiation and holoenzyme isomerization. We infer that these sites in σ54 may be involved in interactions with the core that contribute to maintenance of alternative states of the holoenzyme needed for either the stable closed promoter complex conformation or the isomerized holoenzyme conformation associated with the open promoter complex. One site of σ54 proximity to the core is apparently not evident with σ70, and may represent a specialized interaction. [ABSTRACT FROM AUTHOR]

Background Influenza virus RNA polymerase with the subunit composition of PB1-PB2-PA is a unique multifunctional enzyme with the activities of both synthesis and cleavage of RNA, and is involved in both transcription and replication of the RNA genome. Transcription is initiated by using capped RNA fragments, which are generated after cleavage of host cell mRNA by the RNA polymerase-associated capped RNA endonuclease. To identify the RNA cap 1-binding site on the RNA polymerase, viral ribonucleoprotein (RNP) cores were subjected to UV-crosslinking with RNA which was labelled with 32P only at the cap-1 structure. Results After SDS-PAGE of UV-crosslinked cores, 32P was found to be associated only with the PB2 subunit (759 amino acid residues). The labelled PB2 was subjected, together with PB2 expressed in E. coli, to limited digestion with V8 protease. Analysis of the amino terminal sequences of some isolated fragments with the crosslinked cap-1 indicated that two separate sequences within the PB2 were involved in RNA cap-1 binding, one (N-site) at the N-terminal proximal region approximately between amino acid residues 242–282 downstream from the PB1 subunit-binding site and the other (C-site) between residues 538–577 including the cap-binding motifs. Two lines of evidence support the prediction of the involvement of two separate PB2 sequences on the RNA cap-binding: (i) cross-linking of the capped RNA on to expressed and isolated PB2 fragments, each containing either the N-site or the C-site; and (ii) competition of capped RNA-binding to PB2 by both of the N- and C-terminal PB2 fragments. Taking together, we propose that two separate sequences within PB2 constitute the capped RNA-binding site of the RNA polymerase. Conclusion Two separate sequences, one N-(242–282) and the other C-terminal (538–577) proximal segments of PB2 subunit, constitute the RNA cap-binding site of the influenza virus RNA polymerase. [ABSTRACT FROM AUTHOR]

The σS subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and is required for promoter recognition of many stationary phase genes. We have analysed open complexes of EσS RNA polymers, using σS derivatives carrying single cysteine residues at nine different positions to which the reagent FeBABE has been tethered. All holoenzymes but one formed transcriptionally active open complexes at three different promoters (osmY, galP1 and lacUV5). The chemical nuclease FeBABE can cleave DNA in proximity to the chelate. The overall cutting pattern of EσS open complexes does not depend on the nature of the promoter and is similar to that obtained with Eσ70, but extends towards the downstream part of the promoter. The strongest cleavages are observed with FeBABE positioned on cysteines in regions 2.2 to 3.1. In contrast to σ70 region 2.1 of σS appears to be far from DNA. Region 4.2 of σS appears less accessible than its counterpart in σ70 and FeBABE positioned in the turn of the helix-turn-helix (HTH) motif in region 4.2 reacts only weakly with the −35 promoter element. This provides a structural basis for the minor role of the −35 sequence in σS-dependent promoter recognition. [ABSTRACT FROM AUTHOR]

Studies the genetics and related fields of research on bacterial RNA polymerases (RPase) particularly in Escherichia coli. Role of RPase in transcription of genetic information from DNA to RNA in procaryotes and eucaryotes; Nature and mechanism of enzymatic RNA synthesis; Structure, function, synthesis and assembly of the polymerase subunit.

BackgroundInhibition of transcription of rRNA in Escherichia coli upon amino acid starvation is thought to be due to the binding of ppGpp to RNA polymerase. However, the nature of this interaction still remains obscure. ResultsHere, the azido-derivative of ppGpp was synthesized from azido-GDP and [γ-32P]ATP by way of the phosphate transfer reaction of the RelA enzyme. The product was subsequently characterized by one and two-dimensional chromatography. The resulting compound [32P]azido-ppGpp, where the azido group is attached to the base moiety, was purified to homogeneity and was photo-crosslinked to Escherichia coli RNA polymerase. SDS-PAGE analysis of the azido-ppGpp-bound enzyme, tryptic digestion and Western blot analysis suggested that azido-ppGpp binds to the β-subunit of RNA polymerase. ConclusionIt was observed that both the N-terminal and C-terminal domains of the β-subunit were labelled with azido-ppGpp in the native enzyme. However, under denaturing conditions only the C-terminal part from amino acid residue 802 to residue 1211/1216/1223 was predominantly crosslinked to azido-ppGpp. The excess of unlabelled ppGpp competes with azido-ppGpp for binding to the enzyme. azido-ppGpp inhibits single-round transcription at the stringent promoter like rrnBP1. In addition, ribosomal protein genes were also found to be inhibited by N3ppGpp. On the other hand, transcription at the lac UV5 promoter remained unaffected upon the addition of azido-ppGpp. [ABSTRACT FROM AUTHOR]

Immunoblotting of size-separated whole cell proteins permitted the study of protein-protein interaction. Briefly, proteins obtained from cleared cell lysates of Escherichia coli were separated by glycerol gradient centrifugation and analysed by blotting against a set of specific antibodies. We have applied this procedure to the assembly of 11 N-terminal amber fragments of the β subunit of E. coli RNA polymerase ranging in size between 97% and 23% the length of the intact β polypeptide (1342 amino acids). In this way, we have been able to define regions on the β polypeptide involved in the assembly of RNA polymerase. [ABSTRACT FROM AUTHOR]

The carboxy-terminal domain (CTD) of Escherichia coli RNA polymerase α subunit was specifically modified by a reporter label, fluorescein mercuric acetate (FMMA), conjugated to Cys269 on the surface of UP element recognition helix. The modified enzyme was used to investigate RNA polymerase interaction with different promoters, either with or without an UP element. In a single-round transcription assay, the activity of modified RNA polymerase was found to decrease as measured with rrnBP1, trpP and lacP2 promoters but not with many other promoters including mutant rrnBP1 without the UP element, supporting the idea that Cys269 or the domain including Cys269 is involved in UP element recognition. Both trpP and lacP2 have sequence similarity to the rrnBP1 UP element. The chemical modification of RNA polymerase, however, did not affect an apparent equilibrium dissociation constant with rrnBP1, as measured by gel-retardation assays, indicating that the DNA-binding ability is retained even after FMMA conjugation. Interaction with the rrnBP1 UP element led to substantial alterations in the spectral parameters of the reporter label, which are different from those induced by complex formation with promoters without UP elements. A pronounced spectral blue shift suggests that the labeled surface of αCTD closely approaches the charged UP DNA helix. These observations imply that the fluorescent labeling at Cys269 can be used as a good tool for monitoring the presence or absence of an UP element in a given promoter. Spectral parameters of the label displayed the spectral blue shift when the modified RNA polymerase interacted with trpP, supporting the prediction that this promoter carries an rrnBP1-type UP element. [ABSTRACT FROM AUTHOR]

Purified MalE-SoxS fusion protein specifically stimulated in vitro transcription of the Escherichia coli zwf, fpr, fumC, micF, nfo, and soda genes, indicating that activation of the superoxide regulon requires only SoxS. As in vivo, a 21 bp sequence adjacent to the zwf promoter was able to activate transcription of an heterologous promoter in vitro. Activation of zwf and fpr transcription required the C-terminal domain (CTD) of the RNA polymerase alpha subunit, while stimulation of fumC, micF, nfo, and soda transcription was independent of CTD truncation. Thus, like the catabolite gene activator protein (CAP), SoxS is an 'ambidextrous' activator, activation only requiring the α CTD in a subset of regulated promoters. Indeed, the -35 hexamers of the zwf and fpr promoters lie downstream of the respective MalE-SoxS binding sites, while the binding sites of fumC, micF, nfo, and soda overlap their -35 promoter hexamers. [ABSTRACT FROM AUTHOR]

Transcription in vitro of two osmoregulated promoters, for the Escherichia coli osmB and osmY genes, was analysed using two species of RNA polymerase holoenzyme reconstituted from purified core enzyme and either σD (σ70, the major σ in exponentially growing cells) or σS (σ38, the principal σ at stationary growth phase). Under conditions of low ionic strength, the osmB and osmY promoters were transcribed by both EσD and EσS. Addition of up to 400 mM potassium glutamate (K glutamate), mimicking the intracellular ionic conditions under hyper-osmotic stress, specifically enhanced transcription at these promoters by EσS but inhibited that by EσD. At similar high concentrations of potassium chloride (KCl), however, initiation at both these promoters was virtually undetectable. These data suggest that the RNA polymerase, EσS, itself can sense osmotic stress by responding to changes in intracellular K glutamate concentrations and altering its promoter selectivity in order to recognize certain osmoregulated promoters. [ABSTRACT FROM AUTHOR]

The effects of a number of mutations in crp have been measured at different cyclic AMP receptor protein (CRP)-dependent Class II promoters, where the CRP-binding site is centred around 41 1/2 base pairs upstream from the transcription start point. The amino acid substitutions HL159 and TA158 result in reduced CRP-dependent activation, but the reduction varies from one Class II promoter to another. Deletions in the C-terminus of the RNA polymerase alpha subunit suppress the effects of HL159 and TA158. The role of the C-terminus of alpha at these promoters is assessed. Other changes at E58, K52 and E96 affect CRP activity specifically at Class II promoters and their role is discussed. [ABSTRACT FROM AUTHOR]

The N-terminal two-thirds of the α subunit of Escherichia coli RNA polymerase plays an essential role in the initiation of subunit assembly, by gathering two large subunits, β and β', together into a core-enzyme complex. One group of RNA polymerase mutants deficient in response to transcription activation carries mutations in the C-terminal region of the a subunit, indicating that the C-terminal region of the a subunit is involved in protein-protein contact in positive control of transcription. A set of activators (class I transcription factors) which make contact with this contact site I region on RNA polymerase α subunit bind in most cases to DNA upstream of the promoter =35 signal. Genetic fine mapping indicates that a cluster of subsites exists in the contact site I region, each interacting with a set of the class I factors and each consisting of a structure formed by only 5-10 amino acid residues. [ABSTRACT FROM AUTHOR]

The C-terminal region (amino acid residues 236-329) of the Escherichia coli RNA polymerase α subunit carries the contact site 1 for positive transcription factors. For detailed mapping of the contact site for the cAMP receptor protein (CRP), we made a library of mutant rpoA by polymerase chain reaction (PCR) mutagenesis, such that each should carry a single mutation on average and exclusively in the C-terminal half of the rpoA gene, and then screened this library for mutants with decreased expression of the lacZ gene. Reconstituted holoenzyme containing the mutant α subunits transcribed galP1 but not lacP1 in vitro in the presence of cAMP-CRP. DNA sequence determination of several 'Lac-' mutant rpoA genes revealed that all had mutations clustered within a short segment near the C-terminus of α, between amino acid residues 265 and 270. A cluster of contact sites appear to exist within the contact site I region, each comprising of about five amino acids and responding in molecular communication with a different transcription factor(s). [ABSTRACT FROM AUTHOR]

Influenza virus RNA polymerase is a multifunctional and multisubunit enzyme consisting of three viral P proteins, PB1, PB2, and PA. We have previously shown that radioactive 8-azido GTP (8-N3 GTP) was photo-crosslinked specifically to the PB1 subunit. Here we confirmed the specific crosslinking of PB1 using oxidized GTP and further identified the GTP analogue-binding domains after proteolytic cleavage of the crosslinked PB1 with V8 protease. The cleavage pattern of PB1 was determined by analysis of the amino-terminal proximal sequence of fragments generated in the presence of increasing concentrations of V8 protease. The GTP-crosslinking was identified in three fragments: two adjacent fragments, P6 starting from residue 179 and P11b starting from residue 298; and the third fragment, P11c, starting from residue 458. Thus, we propose that two GTP-binding sites exist in the PB1 subunit, i.e., the amino terminal-proximal site I located at the boundary between P6 and P11b, and the carboxy terminal-proximal site II on P11c fragment. The locations of GTP-binding sites I and II are close to those of sequence motif A and motif D, respectively, conserved among RNA-dependent RNA polymerases. Of the two fragments forming site I, the crosslinking of 8-N3 GTP is higher to P11b, while that of oxidized GTP is higher to P6, suggesting that the ribose and guanine moieties of GTP bound in this binding pocket face P6 and P11c, respectively. From the V8 concentration-dependent change in proteolytic cleavage pattern, it is likely that the two GTP-binding sites on PB1 protein are located on structurally different domains. The existence of two GTP-binding sites is discussed in relation to the binding sites for substrates, primers, and products. [ABSTRACT FROM AUTHOR]

Influenza virus RNA polymerase is composed of three viral P proteins (PB 1, PB2, and PA) and involved in both transcription and replication of the viral RNA genome. The catalytic site for RNA polymerization is located on the PB 1 subunit. To identify the primer ATP-binding site, we have employed a highly selective cross-linking technique: three structurally diverse ATP analogues with reactive groups on their phosphate moieties were first cross-linked to the viral RNA polymerase, and the cross-linked nucleotides were then elongated to dinucleotides by adding the second substrate [α-32P]GTP. Only the ATP analogues tethered to the primer-binding site could be elongated to radioactive AG dinucleotides. Using this catalytically competent cross-linking procedure, the PB 1 subunit was found to be the only labeled subunit. Limited proteolysis of the labeled PB1 by VS protease revealed the segment between amino acids 179 and 297 as the only bearer of the radioactive label. Thus, we concluded that this region of PB 1 faces the 5' end of the primer nucleotide. In support of this prediction, the cross-linked dinucleotides were further elongated up to 8-10 nucleotides in length upon addition of all four substrates. This result suggests that the substantial mass of RNA can be accommodated between the binding site for the primer (and nascent RNA) and the catalytic center of RNA polymerization. [ABSTRACT FROM AUTHOR]

The σ[sup 54] promoter specificity factor is distinct from σ[sup 70]-type factors. The σ[sup 54]-RNA polymerase binds to promoters with conserved sequence elements at -24 and -12 and utilizes specialized enhancer-binding activatots to convert, through an ATP-dependent process, closed promoter complexes to open promoter complexes. The interface between σ[sup 54]-RNA polymerase and promoter DNA is poorly characterized, contrasting with σ[sup 70]. Here, σ[sup 54] was modified with strategically positioned cleavage reagents to provide physical evidence that the highly conserved RpoN box motif of σ[sup 54] is close to and may therefore interact with the consensus -24 promotet element. We show that the spatial relationship between the σ[sup 54]-RNA polymerase and the -24 promoter element remains unchanged during closed to open complex conversion and transcription initiation but changes during the early elongation phase. In contrast, the spatial relationship between σ[sup 54]-RNA polymerase and the consensus -12 promoter element changes upon conversion of the closed promoter complex to an open one. We provide evidence that some -12 promoter region-σ[sup 54] interactions are dependent upon either the core RNA polymerase or a fork junction DNA structure at the -12-position, indicating that DNA fork junctions can substitute for core RNAP. We also show the β-subunit flap domain contributes to different sets of σ-prorooter DNA interactions at σ[sup 54]- and σ[sup 70]-dependent promoters. [ABSTRACT FROM AUTHOR]

Proposes the mapping of sigma D (Rsd) regulator for the Escherichia coli RNA polymerase sigma subunit. Reduction in omega-dependent gene expression following Rsd overproduction; Role of protein residues in promoting transcription initiation; Complex formation testing between Rsd and Ala-substituted omega subunits.

Presents a study which analyzes the interaction of Rpb3 with other RNA polymerase II subunits by far-Western blot analysis and glutathione Schizosaccharomyces transferase (GST) pull-down assay, while carrying out the mapping of subunit-subunit contact sites on Rpb3. Experimental procedures conducted in the study; Identification of Rpb3-contact subunits: Far-Western blot analysis of a mixture of isolated subunits; Indepth look at the study.

The promoter selectivity of Escherichia coli RNA polymerase is determined by the sigma subunit with promoter recognition activity. The model prokaryote Escherichia coli contains seven species of the sigma subunit, each recognizing a specific set of promoters. The major sigma subunit, sigma-70 encoded by rpoD, plays a major role in transcription of growth-related genes. Concomitant with the increase in detection of promoters functioning in vivo under various stressful conditions, the variation is expanding in the consensus sequence of RpoD promoters. In order to identify the canonical sequence of “constitutive promoters” that are recognized by the RNA polymerase holoenzyme containing RpoD sigma in the absence of supporting transcription factors, an in vitro mixed transcription assay was carried out using a whole set of variant promoters, each harboring one base replacement, within the model promoter with the conserved -35 and -10 sequences of RpoD promoters. The consensus sequences, TTGACA(-35) and TATAAT(-10), were identified to be ideal for the maximum level of open complex formation and the highest rate of promoter opening, respectively. For identification of the full range of constitutive promoters on the E. coli genome, a total of 2,701 RpoD holoenzyme-binding sites were identified by Genomic SELEX screening, and using the reconfirmed consensus promoter sequence, a total of maximum 669 constitutive promoters were identified, implying that the majority of hitherto identified promoters represents the TF-dependent “inducible promoters”. One unique feature of the constitutive promoters is the high level of promoter sequence conservation, about 85% carrying five-out-of-six agreements with -35 or -10 consensus sequence. The list of constitutive promoters provides the community resource toward estimation of the inducible promoters that operate under various stressful conditions in nature. [ABSTRACT FROM AUTHOR]

Abstract: It is known that polyamines increase cell growth through stimulation of the synthesis of several kinds of proteins encoded by the so-called “polyamine modulon”. We recently reported that polyamines also increase cell viability at the stationary phase of cell growth through stimulation of the synthesis of ribosome modulation factor, a component of the polyamine modulon. Accordingly, we looked for other proteins involved in cell viability whose synthesis is stimulated by polyamines. It was found that the synthesis of ppGpp regulatory protein (SpoT) and ω protein of RNA polymerase (RpoZ) was stimulated by polyamines at the level of translation. Stimulation of the synthesis of SpoT and RpoZ by polyamines was due to an inefficient initiation codon UUG in spoT mRNA and an unusual location of a Shine–Dalgarno (SD) sequence in rpoZ mRNA. Accordingly, the spoT and rpoZ genes are components of the polyamine modulon involved in cell viability. Reduced cell viability caused by polyamine deficiency was prevented by modified spoT and rpoZ genes whose synthesis was not influenced by polyamines. Under these conditions, the level of ppGpp increased in parallel with increase of SpoT protein. The results indicate that polyamine stimulation of synthesis of SpoT and RpoZ plays important roles for cell viability through stimulation of ppGpp synthesis by SpoT and modulation of RNA synthesis by ppGpp–RpoZ complex. [Copyright &y& Elsevier]

A heterodimer formed by the A14 and A43 subunits of RNA polymerase (pol) I in Saccharomyces cerevisiae is proposed to correspond to the Rpb4/Rpb7 and C17/C25 heterodimers in pol II and pol III, respectively, and to play a role(s) in the recruitment of pol I to the promoter. However, the question of whether the A14/A43 heterodimer is conserved in eukaryotes other than S. cerevisiae remains unanswered, although both Rpb4/Rpb7 and C17/C25 are conserved from yeast to human. To address this question, we have isolated a Schizosaccharomyces pombe gene named ker1+ using a yeast two-hybrid system, including rpa21+, which encodes an ortholog of A43, as bait. Although no homolog of A14 has previously been found in the S. pombe genome, functional characterization of Ker1p and alignment of Ker1p and A14 showed that Ker1p is an ortholog of A14. Disruption of ker11+ resulted in temperature-sensitive growth, and the temperature-sensitive deficit of ker1&Δ was suppressed by overexpression of either rpa21+ or rrn3+, which encodes the rDNA transcription factor Rrn3p, suggesting that Ker1p is involved in stabilizing the association of RPA21 and Rrn3p in pol I. We also found that Ker1p dissociated from pol I in post-logphase cells, suggesting that Ker1p is involved in growth-dependent regulation of rDNA transcription. [ABSTRACT FROM AUTHOR]

The interactions between the σ[sup 54]-containing RNA polymerase (σ[sub 54]-RNAP) and the region of the Pseudomonas putida Pu promoter spanning from the enhancer to the binding site for the integration host factor (IHF) were analyzed both by DNase I and hydroxyl radical footprinting. A short Pu region centered at position -104 was found to be involved in the interaction with σ[sup 54]RNAP, both in the absence and in the presence of IHF protein. Deletion or scrambling of the -104 region strongly reduced promoter affinity in vitro and promotet activity in vivo, respectively. The reduction in promoter affinity coincided with the loss of IHF-mediated recruitment of the σ[sup 54]-RNAP in vitro. The experiments with oriented-α σ[sup 54]-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two α subunit carboxylterminal domains (αCTDs) both at the -104 region and in the surroundings of position -78. The addition of IHF resulted in perfect position symmetry of the two αCTDs. These results indicate that, in the absence of IHF, the σ[sup 54]-RNAP asymmetrically uses only one αCTD subunit to establish productive contacts with upstream sequences of the Pu promoter. In the presence of IHFinduced curvature, the closer proximity of the upstream DNA to the body of the σ[sup 54]-RNAP can allow the other αCTD to be engaged in and thus favor closed complex formation. [ABSTRACT FROM AUTHOR]

Examines the importance of the C-terminal domain of the alpha subunit of RNA polymerase (RNAP) on luxR-dependent transcriptional activation of the lux operon during quorum sensing in Vibrio fischeri. Measurement of the rate of transcription of the lux operon vis luciferase assays; Regulation of gene expression; Purification of variant RNAP holoenzymes.

Presents a study pertaining to the proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase ... subunit. Information on the Escherichia coli RNA polymerase core enzyme; Dimerization of the ... subunit; Materials and methods used; Results from the study.