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The delta subunit of RNA polymerase, RpoE, is widespread in low-G+C Gram-positive bacteria and is thought to play a role in enhancing transcriptional specificity by blocking RNA polymerase binding at weak promoter sites and stimulating RNA synthesis by accelerating core enzyme recycling. Despite the well-studied biochemical properties of RpoE, a role for this protein in vivo has not been defined in depth. In this study, we show that inactivation of rpoE in the human dental caries pathogen Streptococcus mutans causes impaired growth and loss of important virulence traits, including biofilm formation, resistance to antibiotics, and tolerance to environmental stresses. Complementation of the mutant with rpoE expressed in trans restored its phenotype to wild type. The luciferase fusion reporter showed that rpoE was highly transcribed throughout growth and that acid and hydrogen peroxide stresses repressed rpoE expression. Transcriptome profiling of wild-type and ArpoE cells in the exponential and early stationary phase of growth, under acid and hydrogen peroxide stress and under both stresses combined, revealed that genes involved in histidine synthesis, malolactic fermentation, biofilm formation, and antibiotic resistance were downregulated in the ArpoE mutant under all conditions. Moreover, the loss of RpoE resulted in dramatic changes in transport and metabolism of carbohydrates and amino acids. Interestingly, differential expression, mostly upregulation, of 330 noncoding regions was found. In conclusion, this study demonstrates that RpoE is an important global modulator of gene expression in S. mutans which is required for optimal growth and environmental adaptation. doi: 10.1128/JB.00653-10

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5'-phosphorylated viral mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional mRNA capping reaction catalyzed by the RNA:GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to GDP to produce the capped RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the RNA transfer reaction. To locate the active site of the PRNTase domain in the L protein, the covalent RNA attachment site was mapped. We found that the 5'monophosphate end of the RNA is linked to the histidine residue at position 1,227 (H1227) of the L protein through a phosphoamide bond. Interestingly, H1227 is part of the histidine-arginine (HR) motif, which is conserved within the L proteins of the NNS RNA viruses including rabies, measles, Ebola, and Borna disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing antiviral agents. nonsegmented negative-strand RNA virus | polyribonucleotidyltransferase | RNA-dependent RNA polymerase | L protein | mRNA modification doi/10.1073/pnas.0913083107

Fluorescent RNA is an important analytical tool in medical diagnostics, RNA cytochemistry, and RNA aptamer development. We have synthesized the fluorescent ribonucleotide analogue 1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate (tCTP) and tested it as substrate for T7 RNA polymerase in transcription reactions, a convenient route for generating RNA in vitro. When transcribing a guanine, T7 RNA polymerase incorporates tCTP with 2-fold higher catalytic efficiency than CTP and efficiently polymerizes additional NTPs onto the tC. Remarkably, T7 RNA polymerase does not incorporate tCTP with the same ambivalence opposite guanine and adenine with which DNA polymerases incorporate the analogous dtCTP. While several DNA polymerases discriminated against a d(tC-A) base pair only by factors < 10, T7 RNA polymerase discriminates against tC-A base pair formation by factors of 40 and 300 when operating in the elongation and initiation mode, respectively. These catalytic properties make T7 RNA polymerase an ideal tool for synthesizing large fluorescent RNA, as we demonstrated by generating a ~800 nucleotide RNA in which every cytosine was replaced with tC. 10.1021/ac902456n

Primordial organisms of the putative RNA world would have required polymerase ribozymes able to replicate RNA. Known ribozymes with polymerase activity best approximating that needed for RNA replication contain at their catalytic core the class I RNA ligase, an artificial ribozyme with a catalytic rate among the fastest of known ribozymes. Here we present the 3.0 angstrom crystal structure of this ligase. The architecture resembles a tripod, its three legs converging near the ligation junction. Interacting with this tripod scaffold through a series of 10 minor-groove interactions (including two A-minor triads) is the unpaired segment that contributes to and organizes the active site. A cytosine nucleobase and two backbone phosphates abut the ligation junction; their location suggests a model for catalysis resembling that of proteinaceous polymerases. 10.1126/science.1174676

NusA and NusG are transcription elongation factors that bind to RNA polymerase (RNAP) after [sigma] subunit release. Escherichia coli NusA (Nus[A.sub.Ec]) stimulates intrinsic termination and RNA[P.sub.Ec] pausing, whereas NuS[G.sub.Ec] promotes Rho-dependent termination and pause escape. Both Nus factors also participate in the formation of RNA[P.sub.Ec] antitermination complexes. We showed that Bacillus subtilis NusA (NUS[A.sub.Bs]) stimulates intrinsic termination and RNA[P.sub.Bs] pausing at U107 and U144 in the trpEDCFBA operon leader. Pausing at U107 and U144 participates in the transcription attenuation and translational control mechanisms, respectively, presumably by providing additional time for trp RNA-binding attenuation protein (TRAP) to bind to the nascent trp leader transcript. Here, we show that Nus[G.sub.Bs] causes modest pause stimulation at U107 and dramatic pause stimulation at U144. NUS[A.sub.Bs] and Nus[G.sub.Bs] act synergistically to increase the U107 and U144 pause half-lives. NuS[G.sub.Bs]-stimulated pausing at U144 requires RNA[P.sub.Bs], whereas Nus[A.sub.Bs] stimulates pausing of RNA[P.sub.Bs] and RNA[P.sub.Ec] at the U144 and E. coli his pause sites. Although Nus[G.sub.Ec] does not stimulate pausing at U144, it competes with Nus[G.sub.Bs]-stimulated pausing, suggesting that both proteins bind to the same surface of RNA[P.sub.Bs]. Inactivation of nusG results in the loss of RNAP pausing at U144 in vivo and elevated trp operon expression, whereas plasmid-encoded NusG complements the mutant defects. Overexpression of nusG reduces trp operon expression to a larger extent than overexpression of nusA. transcription attenuation | translational control | NusA

The transcription initiation and elongation steps of protein-coding genes usually rely on unrelated protein complexes. However, the TFIIS elongation factor is implicated in both processes. We found that, in the absence of the Med31 Mediator subunit, yeast cells required the TFIIS polymerase II (Pol II)-binding domain but not its RNA cleavage stimulatory activity that is associated with its elongation function. We also found that the TFIIS Pol II-interacting domain was needed for the full recruitment of Pol II to several promoters in the absence of Med31. This work demonstrated that, in addition to its thoroughly characterized role in transcription elongation, TFIIS is implicated through its Pol II-binding domain in the formation or stabilization of the transcription initiation complex in vivo. RNA polymerase II | Saccharomyces cerevisiae | transcription regulation | Med31 | Mediator subunit

Terminal RNA uridylyltransferases (TUTases) catalyze template-independent UMP addition to the 3' hydroxyl of RNA. TUTases belong to the DNA polymerase [beta] superfamily of nucleotidyltransferases that share a conserved catalytic domain bearing three metal-binding carboxylate residues. We have previously determined crystal structures of the UTP-bound and apo forms of the minimal trypanosomal TUTase, TbTUT4, which is composed solely of the N-terminal catalytic and C-terminal base-recognition domains. Here we report crystal structures of TbTUT4 with bound CTP, GTP, and ATP, demonstrating nearly perfect superposition of the triphosphate moieties with that of the UTP substrate. Consequently, at physiological nucleoside 5'triphosphate concentrations, the protein-uracil base interactions alone are not sufficient to confer UTP selectivity. To resolve this ambiguity, we determined the crystal structure of a prereaction ternary complex composed of UTP, TbTUT4, and UMP, which mimics an RNA substrate, and the postreaction complex of TbTUT4 with UpU dinucleotide. The UMP pyrimidine ring stacks against the uracil base of the bound UTP, which on its other face also stacks with an essential tyrosine. In contrast, the different orientation of the purine bases observed in cocrystals with ATP and GTP prevents this triple stacking, precluding productive binding of the RNA. The 3' hydroxyl of the bound UMP is poised for in-line nucleophilic attack while contributing to the formation of a binding site for a second catalytic metal ion. We propose a dual role for RNA substrates in TUTase-catalyzed reactions: contribution to selective incorporation of the cognate nucleoside and shaping of the catalytic metal binding site. crystal structure | nucleotidyl transferase | RNA editing | Trypanosoma | terminal RNA uridylyltransferase poly(A) polymerase

Single-molecule FRET is a powerful tool for probing the kinetic mechanism of a complex enzymatic reaction. However, not every reaction intermediate can be identified via a distinct FRET value, making it difficult to fully dissect a multistep reaction pathway. Here, we demonstrate a method using sequential kinetic experiments to differentiate each reaction intermediate by a distinct time sequence of FRET signal (a kinetic 'fingerprint'). Our model system, the two-way junction hairpin ribozyme, catalyzes a multistep reversible RNA cleavage reaction, which comprises two structural transition steps (docking/undocking) and one chemical reaction step (cleavage/ligation). Whereas the docked and undocked forms of the enzyme display distinct FRET values, the cleaved and ligated forms do not. To overcome this difficulty, we used [Mg.sup.2+] pulse--chase experiments to differentiate each reaction intermediate by a distinct kinetic fingerprint at the single-molecule level. This method allowed us to unambiguously determine the rate constant of each reaction step and fully characterize the reaction pathway by using the chemically competent enzyme-substrate complex. We found that the ligated form of the enzyme highly favors the docked state, whereas undocking becomes accelerated upon cleavage by two orders of magnitude, a result different from that obtained with chemically blocked substrate and product analogs. The overall cleavage reaction is rate-limited by the docking/undocking kinetics and the internal cleavage/ligation equilibrium, contrasting the rate-limiting mechanism of the four-way junction ribozyme. These results underscore the kinetic interdependence of reversible steps on an enzymatic reaction pathway and demonstrate a potentially general route to dissect them. fluorescence resonance energy transfer | hairpin ribozyme | reaction kinetics | ribozyme

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.04.034 Byline: Shanil P. Haugen (1), Melanie B. Berkmen (1), Wilma Ross (1), Tamas Gaal (1), Christopher Ward (1), Richard L. Gourse (1) Abstract: Regulation of transcription initiation is generally attributable to activator/repressor proteins that bind to specific DNA sequences. However, regulators can also achieve specificity by binding directly to RNA polymerase (RNAP) and exploiting the kinetic variation intrinsic to different RNAP-promoter complexes. We report here a previously unknown interaction with Escherichia coli RNAP that defines an additional recognition element in bacterial promoters. The strength of this sequence-specific interaction varies at different promoters and affects the lifetime of the complex with RNAP. Selection of rRNA promoter mutants forming long-lived complexes, kinetic analyses of duplex and bubble templates, dimethylsulfate footprinting, and zero-Angstrom crosslinking demonstrated that I subunit region 1.2 directly contacts the nontemplate strand base two positions downstream of the -10 element (within the 'discriminator' region). By making a nonoptimal I1.2-discriminator interaction, rRNA promoters create the short-lived complex required for specific responses to the RNAP binding factors ppGpp and DksA, ultimately accounting for regulation of ribosome synthesis. Author Affiliation: (1) Department of Bacteriology, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706, USA Article History: Received 14 January 2006; Revised 16 March 2006; Accepted 11 April 2006 Article Note: (miscellaneous) Published: June 15, 2006

Fluorescence resonance energy transfer (FRET) is used to investigate the conformation of promoter DNA in the closed and open complexes of T7 RNA polymerase (T7 RNA P). It is proposed that promoter opening occurs spontaneously upon DNA bending and/or untwisting as free energy is gained through interactions of the melted promoter with the T7 RNAP active site.

Elucidation of the temporal coupling in DNA binding, bending, and opening processes that occur during initiation is presented. The results provided are consistent with the mechanism in which the initial binding of T7 RNAP to the promoter results in a closed complex, which is then converted into an open complex in which promoter is both sharply bent and melted.

The DNA damage stall RNA polymerase (RNAP), depending on sequence context, the conformation of the base when it is met by RNAP and the presence of DNA damage-binding proteins. Transcription-coupled repair (TCR) recognizes and removes the stalled RNAP, repairs and resets transcription allowing RNA synthesis recovery, while transcriptional mutagenesis (TM) leads to bypass of the lesions by RNAP and insertion of incorrect nucleotides, leading to generation of a mutant transcript.

A study was conducted to gain insight into the role of the influenza RNA-dependent RNA polymerase (RdRp) virus in regulation of the relative amounts of mRNA transcription and replication of flu RNA genome. The data obtained provides insight into the regulation of RdRp throughout the viral life cycle and its role in synthesizing the appropriate concentration amounts of viral mRNA and replication products (vRNA and cRNA).

Fluorescence resonance energy transfer and hydrodynamic measurements in Escherichia coli [[sigma].sup.32] were assessed to study dynamic features of two alternative sigma factors. Flexibility observed for the free alternative sigma factors which exhibited equilibrium between two conformations, the promoter interacting motifs trapped in the wrong conformation and another less abundant one with a more open and flexible conformation might be important for promoter recognition and interaction with many partner proteins.

NMR studies with [methyl-[super 13C]]methionine-labeled protein are used for comparing the solution structure and dynamics of wild-type and a single site mutation (Gly64Ser) of the poliovirus RNA-dependent RNA polymerase (3[D.sup.pol]). The studies have shown that there is a long-distance network operating throughout 3[D.sup.pol] that has coordinated ligand binding, conformational changes and catalysis and the mutation of Gly64 has resulted in structural and dynamic changes to the network that might affect polymerase fidelity.

The synthesis of streptolydigin is described, which is assembled in a highly convergent and fully stereocontrolled fashion with a longest linear sequence of 24 steps starting from precursors. The assembly process involved the preparation of fully elaborated streptolic and ydiginic subunits of the natural products, followed by a union in a three-step one-pot procedure, which has included Dieckman cyclization with a concomitant imide opening, Horner-Wadsworth-Emmons olefination, and desilylation.

Fleas collected from rodents in the Negev Desert, southern Israel, are molecularly screened for Bartonella species, where two Bartonella gene fragments, corresponding to RNA polymerase B and citrate synthase (gltA), are targeted. The gltA analysis has shown that Bartonella genotypes are highly similar or identical to strains detected in rodent species from different parts of the world.

The studies have shown that in poly(ADP-ribose) glycohydrolase (PARG) null embryonic trophoblast stem (TS) cells that the absence of PAR hydrolysis led to PAR-modified histones H1, H2A and H2B. It is shown that greater DNA accessibility, increased DNA damage, and increased cell death have contributed to the PARG null cell phenotype in response to genotoxic stress.

Sedimentation equilibrium, dynamic light scattering, electrophoretic mobility shift, and transcription activity measurements are used to assess affinity and function of site-specific AsiA mutants which are T4 bacteriophage early gene product that regulates transcription of host and viral genes. The results defined the core interfacial residues of AsiA, establish roles for many of the interfacial amino acids are in agreement with the tenets underlying protein-protein interactions and interfaces, and could provide insight into the mechanistic related to the bacterial RNA polymerase regulation.

Laser tweezers are used to study the structures formed in 5'-[(TGTCCCCACACCCC).sub.2], a predominate variant in the insulin-linked polymorphic region (ILPR) and the formation of i-motif in the cytosine (C)-rich region complementary to G quadruplex-forming sequences. The similar rupture forces of 22-26 pN shared by the i-motif and the partially folded structure which are higher than those that can stall transcription catalyzed by RNA polymerases suggested that either of the structures could stop RNA transcription.

A new genetic tool was designed for in situ detection of plasmid transfer under anaerobic and aerobic conditions. The dual reporter system is based on the T7 RNA polymerase gene and a T7 promoter-driven oxygen-independent green fluorescent protein, evoglow, alone or in combination with red fluorescent protein DsRed.

The processing of site-specific Pt-DNA cross-links in live mammalian cells were explored to gain better insight into mechanism of action of platinum-based anticancer drugs. Cells with either competent or deficient nucleotide excision repair systems were utilized to demonstrate that Pt-DNA cross-links impede transcription by blocking passage of the RNA polymerase complex and that nucleotide excision repair could remove the block and restore transcription.

A study was conducted to probe the effects of Na(+) salts of Glu(-), F(-), and Cl(-) on each step in the critical process related to large-scale conformational changes in RNA polymerases (RNAPs) to form the transcriptionally competent open complex [RP.sub.o]. Based on the analogy to Hofmeister salt effects on protein folding it was proposed that excluded anions Glu(-) and F(-) drive the folding and assembly of the RNAP clamp/jaw domains in the conversion of [I.sub.2] to [RP.sub.o], while Cl(-) does not.

The model T7 RNA polymerase was used to demonstrate the association of reconstituted nucleosomes containing either canonical human recombinant histones or two histone variants, H2A.Z or H3.3 with active transcription. Compared to canonical and H3.3-containing nucleosomes, H2A.Z-containing nucleosomes are refractive to transcription, with residual levels of transcription determined by the sequence of the underlying DNA template.

The RNA polymerases (RNAP)-F/E subcomplex is described, which has formed a stable heterodimer that has bound the nascent RNA and has stimulated the processivity of elongation complexes. The pulsed-EPR method DEER and fluorescence spectroscopy have shown that, upon binding of RNA, F/E has remained in a stable conformation, suggesting that it has served as a structurally rigid guiding rail for the growing RNA chain during transcription.

A convergent strategy with longest linear sequence of 23 steps was used to achieve a highly stereoselective joint total synthesis of the potent polyketide macrolide antibiotics etnangien and etnangien methyl ester. The expedient and flexible strategy of the etnangiens could be adapted to design analogues of these RNA-polymerase inhibitors and allow further exploration of the promising biological potential of these macrolide antibiotics.

A study was conducted to understand the role of the first coiled coil segment of the noncanonical sequence composition for the interaction with cyclin T1 and the second segment which is required to keep the cyclin T-binding domain dimeric upon binding. Analytical results for ultracentrifugation and cross-linking experiments suggested that binding to cyclin T1 induced an asymmetry or sterical hindrance in the first coiled coil segment of dimeric Hexim1 that disallowed formation of a 2:2 complex.

The functional expression of a multisubunit complex on magnetosomes is obtained by examining the display of a chimeric bacterial RNAse P enzyme composed of the protein subunit (C5) of Escherichia coli RNase P and the endogeneous RNA subunit by expressing a translational fusion of C5 with MamC, a known magnetosome protein, in the bacterium Magnetospirillum gryphiswaldense. The results have shown that magnetosomes are used as scaffolds for the display of multisubunit complexes.

The article discusses a new approach, which is used for the first total synthesis of etnangien, the potent RNA-polymerase inhibitor. The method is shown to be highly beneficial, as it leads to high stereoselectivity and yield.

New methods were developed to monitor carbonyl formation on proteins as a function of distance from a hydroxyl generator, iron-(S)-1-[p-(bromoacetamido)benzyl]EDTA (FeBABE), conjugated to an engineered cysteine residue. Intraprotein oxidation of single-cysteine mutants of Escherichia coli [sigma]70 by tethered FeBABE conjugates in complexes with the RNA polymerase core enzyme provide fundamental information related to protein-protein interactions and interpretation of protein footprinting experiments in other systems.

The studies related to synthesis and biological properties of the locked nucleic acid (LNA)-modified cap analogue, [m.sup.7(LNA)]G[5']ppp[5']G, an efficient substrate for T7 RNA polymerase, and the mRNA transcribed, with a poly(A) tail are reported. The findings established the new antireverse cap analogue [m.sup.7](LNA)G[5']ppp[5']G as a potential candidate for RNA-based therapeutic vaccine production as well as studying biochemical processes.

A molecular complex consisting of a DNA template and associating RNA polymerases (RNAPs) has displayed chemokinetic motion driven by transcription substrates nucleoside triphosphates (NTPs). The molecular complex has displayed a biased migration into a concentration gradient of NTPs, resembling chemotaxis, which is described as Molecular Propulsion, in which RNAP transcriptional actions have deformed DNA template conformation engendering measurable enhancement of motility.

The unnatural base pairs d5SICS:dMMO2 and d5SICS:dNaM are selectively transcribed by T7 RNA polymerase and the efficiency of d5SICS:dNaM transcription in both possible strand contexts is marginally reduced relative to that of a natural base pair. The results have shown that d5SICS:dNaM is both replicated and transcribed with efficiencies and fidelities that is sufficient for use as part of an in vitro expanded genetic alphabet.

The first biochemical characterization of XPB, the largest subunit of the eukaryotic transcription factor TFIIH, which is essential for both initiation of transcription by RNA polymerase II and nucleotide excision repair (NER) from bacteria, Mycobacterium tuberculosis and Kineococcus radiotolerans is reported. The ATP hydrolytic acttivity by the bacterial XPBs is strongly dependent on both the nature of a nucleotide triphosphate and that of a divalent metal.

The construction, engineering, and detailed characterization of an artificial genetic AND gate based on the [P.sub.lux1] quorum-sensing promoter and the lac repressor for artificial genetic circuits that could integrate intercellular signaling molecules and commonly used inducing agents is described. The results obtained for the responses of the AND gate offer insight into interaction of the LuxR, LacI, and RNA polymerase to activate gene expression.

The organization of the RNA polymerase (RNAP) catalytic center, which has served as a robust, molecular engine that drives and controls the rate, processivity and fidelity of transcription is discussed.

The experimental studies related to the ability of bacteria to survive environmental stresses are described, which have confirmed the hypothesis that accumulation of a simple signaling molecule, glutamate, could reprogram RNA polymerase in vitro without the need for specific protein receptors. The data obtained demonstrated that glutamate could assist activation of transcription by remodeling the [sigma]38 transcription complex by virtue of acting as a Hofmeister salt.

The studies of antigene locked nucleic acids (agLNAs) that reduce RNA levels of targeted genes block RNA polymerase and transcription factor association at gene promoters, and bind to chromosomal DNA are presented. The data obtained suggested that the mechanism for agLNA-mediated inhibition of gene expression involved binding of LNAs to chromosomal DNA.

Several NMR measurements are conducted to explain the stereochemical determination and complex biosynthetic assembly of etnangien, a highly potent RNA polymerase inhibitor from the myxobacterium Sorangium cellulosum. The biosynthetic gene cluster is shown to exhibit a highly unusual and complex trans-AT type polyketide biosynthesis.

A nucleotide analogue interference mapping (NAIM) was conducted to understand the use of a series of fluorine substituted adenosine analogues that produce large changes in N1 p[K.sub.a] values with minimal structural perturbation. Each of the analogues is found to be readily incorporated by T7 RNA polymerase.

A synthetic method to prepare the initiator nucleotide with the levulinic amide substrate and the photocleavable linker separated by a triethylene glycole spacer is reported. The results showed that small molecule functionalities attached to the phosphate of guanosine monophosphate via a photocleavable linker moiety could be conveniently attached to RNA molecules of a broad size range via transcription priming using T7 RNA polymerase.

Shuttle vectors that replicate stably and express selectable phenotypes in both Thermococcus kodakaraensis and Escherichia coli are constructed. Affinity purification has confirmed that an hemagglutinin (HA)- plus six-histidine-tagged RpoL subunit (RpoL-HA-[his.sub.6]) synthesized ectopically from a shuttle vector is assembled in vivo into RNA polymerase (RNAP) holoenzymes that are active and is purified directly from Thermococcus kodakaraensis cell lysates by [Ni.sup.2+] binding and imidazole elution.