RNA polymerase

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-s 54 holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAAþ domain and the interdomain linker that connects the AAAþ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAAþ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30-45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.

Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA "scrunching"--in which RNA polymerase (RNAP) remains stationary and unwinds and pulls into itself downstream DNA--that scrunching requires RNA synthesis, and that scrunching depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support existence of an obligatory stressed intermediate, with ~1 turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape.

A gene (pueA, polyurethane esterase A) encoding an extracellular polyurethanase (PueA) was cloned from Pseudomonas chlororaphis into Escherichia coli. The enzyme secreted from E. coli showed esterase activity when assayed with p-nitrophenyl acetate. Subcloning of a 3.2-kb SalI^EcoRI fragment into a T7 RNA polymerase expression vector (pT7-6) produced a 35 S-labeled protein of 65 kDa. Nucleotide sequencing of pueA showed an open reading frame encoding a 65-kDa protein of 617 amino acid residues, with the serine hydrolase consensus sequence GXSXG. PueA was over-expressed using the pT7-6 vector transformed into E. coli BL21(DE3) and was purified in one step using Sephadex G-75. ß : S 0 3 7 8 -1 0 9 7 ( 0 0 ) 0 0 0 9 4 -X

Many flaviviruses are significant human pathogens. The plus-strand RNA genome of a flavivirus contains a 5′ terminal cap 1 structure (m 7 GpppAmG). The flavivirus encodes one methyltransferase (MTase), located at the N-terminal portion of the NS5 RNA-dependent RNA polymerase (RdRp). Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development. The NS5 MTase catalyzes both guanine N7 and ribose 2′-OH methylations during viral cap formation. Representative flavivirus MTases, from dengue, yellow fever, and West Nile virus (WNV), sequentially generate GpppA → m 7 GpppA → m 7 GpppAm. Despite the existence of two distinct methylation activities, the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2′-O methyl groups from SAM during the sequential reactions. Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2′-OH on the first transcribed nucleotide. Mutant enzymes with different methylation defects can trans complement one another in vitro, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro. In the context of the infectious virus, defects in both methylations, or a defect in the N7 methylation alone, are lethal to WNV. However, viruses defective solely in 2′-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel and promising target for flavivirus therapy.

Gene "7" of Escherichia coli phage K1E was proposed to encode a novel DNA-dependent RNA polymerase (RNAP). The corresponding protein was produced recombinantly, purified to apparent homogeneity via affinity chromatography, and successfully employed for in vitro RNA synthesis. Optimal assay conditions (pH 8, 37°C, 10 mM magnesium chloride and 1.3 mM spermidine) were established. The corresponding promoter regions were identified on the phage genome and summarized in a sequence logo. Surprisingly, next to K1E promoters, the SP6 promoter was also recognized efficiently in vitro by K1E RNAP, while the T7 RNAP promoter was not recognized at all. Based on these results, a system for high-yield in vitro RNA synthesis using K1E RNAP was established. The template plasmid is a pUC18 derivative, which enables blue/white screening for positive cloning of the target DNA. Production of more than 5 μg of purified RNA per microgram plasmid DNA was achieved. Finally, in vivo protein production systems for Bacillus megaterium were established based on K1E and SP6 phage RNAP transcription. Up to 61.4 mg g CDW −1 (K1E RNAP) of the reporter protein Gfp was produced in shaking flask cultures of B. megaterium.

The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the X PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.

Many vertebrate small nuclear RNA gene promoters contain an SPH motif in their distal control regions that can confer transcriptional stimulation by RNA polymerase II or RNA polymerase III. Using the human U6 gene SPH motif as a probe, we isolated a cDNA encoding human SPH-binding factor (hSBF) from a HeLa cell expression library. The coding region of hSBF is almost identical to ZNF143, a 626 amino acid, seven zinc finger protein of previously unknown function. Furthermore, the predicted amino acid sequence of hSBF is highly homologous to Xenopus laevis and mouse Staf proteins, that bind to SPH motifs and stimulate transcription of selenocysteine tRNA gene promoters. Recombinant hSBF expressed in vitro or from Escherichia coli bound specifically to the human U6 gene SPH motif as shown by DNase I footprinting and electrophoretic mobility shift assays using various mutant SPH sites as competitors. Antibodies prepared against recombinant hSBF inhibited assembly of native SBF-DNA complexes. Immunodepleted HeLa S100 transcription extract no longer supported elevated levels of transcription by RNA polymerase III from a U6 promoter containing an SPH motif, whereas addition of recombinant hSBF protein to the immunodepleted extract reconstituted stimulated transcription.

In bacteria, initiation of transcription depends on the RNA polymerase subunit, which brings catalytically proficient RNA polymerase core to promoters by binding to specific DNA elements located upstream of the transcription start point. Here, we study -dependent synthesis of a transcript that is used to prime replication of the single-stranded genome of bacteriophage M13. We show that, in this system, plays no role in DNA recognition, which is accomplished solely through RNA polymerase core interaction with DNA downstream of the transcription start point. However, is required for full-sized transcript synthesis by allowing RNA polymerase core to escape into productive elongation. RNA polymerase may play a similar role during replication primer synthesis in other bacterial mobile elements whose life cycle involves a single-stranded DNA stage.

Allicin, one of the active principles of freshly crushed garlic homogenates, has a variety of antimicrobial activities. Allicin in its pure form was found to exhibit i) antibacterial activity against a wide range of Gram-negative and Gram-positive bacteria, including multidrug-resistant enterotoxicogenic strains of Escherichia coli; ii) antifungal activity, particularly against Candida albicans; iii) antiparasitic activity, including some major human intestinal protozoan parasites such as Entamoeba histolytica and Giardia lamblia; and iv) antiviral activity. The main antimicrobial effect of allicin is due to its chemical reaction with thiol groups of various enzymes, e.g. alcohol dehydrogenase, thioredoxin reductase, and RNA polymerase, which can affect essential metabolism of cysteine proteinase activity involved in the virulence of E. histolytica. © Elsevier, Paris alliinase / allicin / thiol enzymes / antibiotics / garlic / antimicrobials * Correspondence and reprints Figure 1. Generation of allicin in a garlic clove.

The combined structural study of proteins and of their corresponding genes utilizing the methods of both protein and nucleotide chemistry greatly accelerates and considerably simplifies both the nucleotide and protein structure determination and, in particular, enhances the reliability of the analysis. This approach has been successfully applied in the primary structure determination of the fl and j' subunits of Escherichia coli DNAdependent RNA potynierase and of their structural genes, yielding a continuous nucleotide sequence (4714 base pairs) that embraces the entire rpoB gene, the initial part of the rpoC gene and the intercistronic region, together with the total amino acid sequence of the fl subunit, comprising 1342 residues, and the N-terminal sequence of the jl' subunit (1 76 residues).

Glossary cAMP: cyclic adenosine monophosphate. CAP: catabolite activator protein. F(bridge)-helix and G(trigger)-loop: mobile elements of the RNAP catalytic center (sometimes also called 'bridge helix' and 'trigger loop', respectively). i+1: the substrate binding site in the RNAP catalytic center. P lac : promoter of the lactose operon. ppGpp: guanosine tetraphosphate, the effector of the stringent response. RNAP: RNA polymerase. RPc: RNAP-promoter closed complex. RPitc: RNAP-promoter initial transcribing complex. RPo: RNAP-promoter open complex. aCTD: carboxy-terminal domain of alpha subunit. s1, s2, s3, s4: evolutionarily conserved domains of s 70 . s3-s4 linker: sigma subunit linker element connecting domains s3 and s4. s 54 : sigma N, the sigma factor regulating nitrogen metabolism. s 70 : sigma 70, the housekeeping sigma factor in Escherichia coli.

Suivant l'initiation de la transcription, l'ARN polymérase T7 rentre immédiatement dans une phase durant laquelle la dissociation du complexe tertiaire enzyme-promoteur-ARN, compétitionne de façon énergétique avec le processus d'élongation, un mécanisme appelé abortive cycling. Il a été démontré que ceci diminue la vitesse d'élongation (1). En introduisant une mutation à point semi-conservatrice, Y659F, dans la séquence codante de l'ARN polymérase T7, nous avons confirmé l'importance de ce résidu dans l'activité enzymatique de la polymérase. Le résidu Y650 de la protéine recombinante correspond au résidu Y639 de la protéine sauvage. Un essai enzymatique a été effectué avec l'ARN polymérase T7 de type sauvage (WT) et l'enzyme mutée, le MAT-ARN polymérase Y650F à l'aide d'un thermocycleur à temps réel. Les résultats démontrent que la mutante possède une activité enzymatique 2X plus élevée que le type sauvage. Cela prouve que le résidu Y650 joue un rôle essentiel dans la formation du complexe ainsi que dans le processus d'élongation. De plus, l'édition du groupement hydroxyle fonctionnel chez la Y650 augmente la vitesse de synthèse d'ARNm complète, soit en favorisant son affinité pour la séquences en aval du promoteur ou en promouvant la processivité.

This study provides a mathematical model of T7 RNA polymerase (T7 RNAP) kinetics under in vitro conditions targeted at application of this model to simulation of dynamic transcription performance. A functional dependence of transcript synthesis rate is derived based on: (a) essential reactant concentrations, including T7 RNAP and its promoter, substrate nucleotides, and the inhibitory byproduct inorganic pyrophosphate; (b) a distinction among vector characteristics such as recognition sequences regulating transcription initiation and termination, respectively; and (c) specific properties of the nucleotide sequence including both transcript length and nucleotide composition. Inactivation kinetics showed a half-life of T7 RNAP activity of 50 min under the conditions applied in vitro using the isolated enzyme. Model parameters and their precision are estimated using dynamic simulation and nonlinear regression analysis. The particular novelty of this model is its capability to incorporate linear genomic sequence information for simulation of nonlinear in vitro transcription kinetics.

The extracytoplasmic function (ECF) sigma factors constitute a diverse group of alternative sigma factors that have been demonstrated to regulate gene expression in response to environmental conditions in several bacterial species. Genes encoding an ECF sigma factor of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium smegmatis, designated sigE, were cloned and analyzed. Southern blot analysis demonstrated the presence of a single copy of this gene in these species and in Mycobacterium bovis BCG, Mycobacterium leprae, and Mycobacterium fortuitum. Sequence analysis showed the sigE gene to be highly conserved among M. tuberculosis, M. avium, M. smegmatis, and M. leprae. Recombinant M. tuberculosis SigE, when combined with core RNA polymerase from M. smegmatis, reconstituted specific RNA polymerase activity on sigE in vitro, demonstrating that this gene encodes a functional sigma factor. Two in vivo transcription start sites for sigE were also identified in M. smegmatis and M. bovis BCG. Comparison of wild-type M. smegmatis with a sigE mutant strain demonstrated decreased survival of the mutant under conditions of high-temperature heat shock, acidic pH, exposure to detergent, and oxidative stress. An inducible protective response to oxidative stress present in the wild type was absent in the mutant. The mycobacterial SigE protein, although nonessential for viability in vitro, appears to play a role in the ability of these organisms to withstand a variety of stresses.

Life appears to be a natural property of matter, but the problem of its origin only arose after early scientists refuted continuous spontaneous generation. There is no chance of life arising 'all at once', we need the standard scientific incremental explanation with large numbers of small steps, an approach used in both physical and evolutionary sciences. The necessity for considering both theoretical and experimental approaches is emphasized. After describing basic principles that are available (including the Darwin-Eigen cycle), the search for origins is considered under four main themes. These are the RNA-world hypothesis; potential intermediates between an RNA-world and a modern world via the evolution of protein synthesis and then of DNA; possible alternatives to an RNA-world; and finally the earliest stages from the simple prebiotic systems to RNA. The triplicase/ proto-ribosome theory for the origin of the ribosome is discussed where triples of nucleotides are added to a replicating RNA, with the origin of a triplet code well-before protein synthesis begins. The length of the code is suggested to arise from the early development of a ratchet mechanism that overcomes the problem of continued processivity of an RNA-based RNA-polymerase. It is probable that there were precursor stages to RNA with simpler sugars, or just two nucleotides, but we do not yet know of any better alternatives to RNA that were likely to arise naturally. For prebiotic stages (before RNA) a flow-reactor model is suggested to solve metabolism, energy gradients, and compartmentation simultaneously -thus the intense interest in some form of flow reactor. If an autocatalytic cycle could arise in such a system we would be major steps ahead. The most likely physical conditions for the origin of life require further clarification and it is still unclear whether the origin of life is more of an entropy (information) problem (and therefore high temperatures would be detrimental), rather than a kinetic problem (where high temperatures may be advantageous).

Mammalian RNA polymerase I1 transcription factor IIE (TFIIE) was purified to apparent homogeneity. The activity copurified with polypeptides of 34 and 56 kDa. The 56-kDa subunit was sufficient for low levels of transcription activity in a transcription system reconstituted in vitro with highly purified general transcription factors and RNA polymerase 11. The 34-kDa polypeptide was found to be stimulatory. The native molecular mass of TFIIE, as determined by gel filtration, was estimated to be approximately 200 kDa, suggesting that TFIIE exists in solution as a tetramer composed of two 56-kDa and two 34-kDa polypeptides. Consistent with previous studies demonstrating an interaction of TFIIE with RNA polymerase 11, we found that the entry of TFIIE into the transcription cycle was subsequent to the entry of RNA polymerase 11. Transcription of protein-coding genes in higher eukaryotes is carried out by a multienzymatic complex that includes RNA polymerase I1 and several accessory transcription factors. One class of factors consists of DNA-binding proteins that recognize specific promoter or enhancer elements, through which they regulate gene-or tissue-specific transcription by RNA polymerase I1 (reviewed in . Another group termed general transcription factors act through the core promoter elements (TATA box and initiator motif) and are required for basal levels of specific transcription initiation at all class two promoters (for review, see . The mechanisms by which the general transcription factors determine the position and directionality of transcription initiation and the biochemical events by which they trigger the formation of the first phosphodiester bond by RNA polymerase I1 are not yet understood.

Potassium permanganate (KMnO 4 ) has widely been used in genomic footprinting assays to map unusual gene structures, including the melting DNA block in transcriptional elongation that results from promoter-proximal pausing of RNA polymerase (Pol) II complexes. Although it has been assumed that DNA-bound proteins do not protect underlying nucleic acids from KMnO 4 modifications, we provide evidence herein that this chemical can readily be used to detect nuclear factor loading at a promoter when using optimized conditions. Moreover, by comparing parallel KMnO 4 and dimethylsulfate (DMS) in vivo footprintings, we show that the utilization of KMnO 4 in combination with another chemical probe maximizes the detection of factor occupancy at a DNA regulatory region, thus providing a better opportunity to define the actual profiles of DNA -protein contacts at given genomic sites in living cells.

A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVΔG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVΔG*G. Pseudotype VSV with the Hantaan (VSVΔG*-HTN) or Seoul (VSVΔG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 105 to 106/ml. The infectivity of VSVΔG*-HTN and VSVΔG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with ...

A method for preparative in vitro mRNA synthesis is presented. The method makes it possible to generate several hundred RNA copies per molecule of linearized DNA template. The protocol is applicable to the synthesis of RNAs of differing lengths (from 200 to 3000 bases), and both SP6 and T7 RNA polymerases can be used. The transcription can be scaled up to at least 6–12 ml, yielding 4.5–18 mg of homogeneous biologically active mRNA.

center and nucleic acid binding sites (Korzheva et al., New York, New York 10016 2000; Gnatt et al., 2001). In this model, the two largest 2 BioMaPS Institute for Quantitative Biology and subunits of RNAP hold the EC together by clamping the Department of Physics 8 bp RNA:DNA hybrid and downstream DNA duplex Rutgers University (Nudler, 1999). These interactions allow the EC to slide Piscataway, New Jersey 08854 along DNA and RNA during elongation and reverse 3 Public Health Research Institute translocation (backtracking) (Komissarova and Kashlev, Newark, New Jersey 07103 1997; Nudler et al., 1997). Based on comparative analysis of bacterial and yeast RNAP structures, Kornberg and coworkers proposed an elementary transcription Summary cycle in which bending of the F bridge helix (F bridge) at the 3Ј face of the RNA:DNA hybrid induces transloca-RNA chain elongation is a highly processive and accution of the nucleic acid by one nucleotide within the rate process that is finely regulated by numerous in-RNAP mainframe, while a subsequent relaxation of the trinsic and extrinsic signals. Here we describe a gen-F bridge opens the substrate binding site for the next eral mechanism that governs RNA polymerase (RNAP) complementary NTP (Gnatt et al., 2001). A bent F bridge movement and response to regulatory inputs such as helix has only been observed in bacterial RNAP strucpauses, terminators, and elongation factors. We show tures (Zhang et al., 1999; Vassylyev et al., 2002) and a that E.coli RNAP moves by a complex Brownian ratchet straight F bridge only in the yeast RNAP II structure mechanism, which acts prior to phosphodiester bond (Cramer et al., 2001). However, protein-RNA crosslinking formation. The incoming substrate and the flexible F data obtained using the E. coli EC strongly suggest the bridge domain of the catalytic center serve as two ability of the F bridge to change its conformation within separate ratchet devices that function in concert to the same enzyme (Epshtein et al., 2002).

Newcastle disease virus (NDV) is classi®ed as a member of the superfamily Mononegavirales in the family Paramyxoviridae. This virus family is divided into two subfamilies, the Paramyxovirinae and the Pneumovirinae. In 1993 the International Committee on the Taxonomy of Viruses rearranged the order of the Paramyxovirus genus and placed NDV within the Rubulavirus genus among the Paramyxovirinae. The enveloped virus has a negative sense single-stranded RNA genome of 15,186 kb which codes for an RNA directed RNA polymerase, hemagglutinin±neuraminidase protein, fusion protein, matrix protein, phosphoprotein and nucleoprotein in the 5 ' to 3 ' direction. The virus has a wide host range with most orders of birds reported to have been infected by NDV. Isolates are characterized by virulence in chickens and are categorized into three main pathotypes depending on severity of disease. Lentogenic isolates are of low virulence while viruses of intermediate virulence are termed mesogenic. Highly virulent viruses that cause high mortality in birds are termed neurotropic or viscerotropic velogenic. Velogenic NDV are List A pathogens that require reporting to the Oce of International Epizootics and outbreaks result in strict trade embargoes. The primary molecular determinant for NDV pathogenicity is the fusion protein cleavage site amino acid sequence. Vaccination for NDV is primarily by mass application of live-virus vaccines among commercial poultry. Although protection is measured by presence of antibodies to NDV, vaccinated B-cell depleted chickens are resistant to disease. Consequently, immune protection involves responses that are presently incompletely de®ned. 7

Although more than 30 Escherichia coli promoters utilize the RNA polymerase holoenzyme containing s S (Es S ), and it is known that there is some overlap between the promoters recognized by Es S and by the major E. coli holoenzyme (Es 70 ), the sequence elements responsible for promoter recognition by Es S are not well understood. To define the DNA sequences recognized best by Es S in vitro, we started with random DNA and enriched for Es S promoter sequences by multiple cycles of binding and selection. Surprisingly, the sequences selected by Es S contained the known consensus elements (210 and 235 hexamers) for recognition by Es 70 . Using genetic and biochemical approaches, we show that Es S and Es 70 do not achieve specificity through 'best fit' to different consensus promoter hexamers, the way that other forms of holoenzyme limit transcription to discrete sets of promoters. Rather, we suggest that Es S -specific promoters have sequences that differ significantly from the consensus in at least one of the recognition hexamers, and that promoter discrimination against Es 70 is achieved, at least in part, by the two enzymes tolerating different deviations from consensus. DNA recognition by Es S versus Es 70 thus presents an alternative solution to the problem of promoter selectivity.

Primase and DnaB helicase play central roles during DNA replication initiation and elongation. Both enzymes are drug targets because they are essential, persistent among bacterial genomes, and have different sequences than their eukaryotic equivalents. Myricetin is a ubiquitous natural product in plants that is known to inhibit a variety of DNA polymerases, RNA polymerases, reverse transcriptases, and telomerases in addition being able to inhibit kinases and helicases. We have shown that myricetin inhibits Escherichia coli DnaB helicase according to a mechanism dominated by noncompetitive behavior with a K(i) of 10.0+/-0.5 microM. At physiological ATP concentration, myricetin inhibits E. coli DnaB helicase with an inhibitory concentration at 50% maximal (IC(50)) of 11.3+/-1.6 microM. In contrast, myricetin inhibited E. coli primase at least 60-fold weaker than DnaB helicase and far weaker than any other polymerase.

In Escherichia coli, mutations conferring rifampicin (Rif) resistance map to the rpoB gene, which encodes the 1342-amino acid/3 subunit of RNA p01ymerase. Almost all sequenced Rif R mutations occur within the Rif region, encompassing rpoB codons 500-575. A strong Rif k mutation lying outside the Rif region, which changed Va1146 to Phe was previously reported, but was not recovered in subsequent studies. Here, we used site-directed mutagenesis followed by selection on Rif to search for Rif R mutations in the evolutionarily conserved segment of rpoB around codon 146. Strong Rif R mutations were obtained when Val x46 was mutated, and several weak Rif R mutations were also isolated near position 146. The results define a new, N-terminal cluster of Rif e mutations, in addition to the classical central Rif region.

A hamster cell line resistant to a-amanitine
has been isolated (a-amr, BHK-T6-G1). Cell extracts
of this mutant have an a-amanitine-resistant RNA
polymerase II (nucleosidetriphosphate:,RNA nucleotidyltransferase,
EC 2.7.7.6) activity as shown by DEAEcellulose
column chromatography. This mutation is
dominant in interspecific hybrids with 3T3 mouse cells.
In such hybrids polyoma virus can grow with equal efficiency
in the presence or absence of the drug, thus indicating
that the RNA polymerase of the unsusceptible parental
cell can participate in the correct transcription of the
viral genome.

The yellow lupin leghemoglobin I gene (IH) was cloned in the PET-3a vector. It was overexpressed in Escherichia coli BLZl(DE3)pLysS cells, under the control of T7 RNA polymerase promoter. The recombinant Lb1 protein, containing E. coli derived heme, was purified to homogeneity using ion exchange and gel filtration chromatography. The recombinant Lb1 protein has spectral and immunochemical properties identical to Lb1 isolated from lupin root nodules. The identity between expressed polypeptide and native Lb1 protein was also supported by microsequencing analysis of the N-terminus of the purified recombinant protein.

Background: Many programs have been developed to identify transcription factor binding sites. However, most of them are not able to infer two-word motifs with variable spacer lengths. This case is encountered for RNA polymerase Sigma (σ) Factor Binding Sites (SFBSs) usually composed of two boxes, called -35 and -10 in reference to the transcription initiation point. Our goal is to design an algorithm detecting SFBS by using combinational and statistical constraints deduced from biological observations.

Northwestern Brazil is a major center of diversity for cactaceous plants, most of which are grown as ornamentals. Cactus virus X (CVX), the only virus reported so far from Brazilian cactaceae, is widely spread. In this study we have characterized viruses of Opuntia tuna (OT), Hylocereus undatus (HU) and Schlumbergera truncata (ST) from the State of São Paulo, showing chlorotic spots and mosaic symptoms. Mechanically inoculated Gomphrena globosa reacted with local lesions, except when OT extracts were used as inoculum, whereas Chenopodium amaranticolor showed erratic systemic symptoms following inoculations from HU and ST. Transmission electron microscopy showed flexuous particles ca. 550 nm in length and bundles of virus particles in leaf cells from naturally and experimentally infected plants. Virus particles reacted faintly with an antiserum to CVX. Nucleotide sequences of the RNA dependent RNA polymerase gene obtained from cloned RT-PCR products, revealed the occurrence of Zygocactus virus X (ZyVX) in OT, mixed infection by ZyVX and Schlumbergera virus X (SchVX) in HU, and mixed infection by ZyVX, SchVX and Opuntia virus X (OpVX) in ST. This is the first report of ZyVX, SchVX and OpVX in Brazil.

A previous comparative-genomic study of large nuclear and cytoplasmic DNA viruses (NCLDVs) of eukaryotes revealed the monophyletic origin of four viral families: poxviruses, asfarviruses, iridoviruses, and phycodnaviruses . Common origin of four diverse families of large eukaryotic DNA viruses. J. Virol. 75 (23), 11720-11734]. Here we update this analysis by including the recently sequenced giant genome of the mimiviruses and several additional genomes of iridoviruses, phycodnaviruses, and poxviruses. The parsimonious reconstruction of the gene complement of the ancestral NCLDV shows that it was a complex virus with at least 41 genes that encoded the replication machinery, up to four RNA polymerase subunits, at least three transcription factors, capping and polyadenylation enzymes, the DNA packaging apparatus, and structural components of an icosahedral capsid and the viral membrane. The phylogeny of the NCLDVs is reconstructed by cladistic analysis of the viral gene complements, and it is shown that the two principal lineages of NCLDVs are comprised of poxviruses grouped with asfarviruses and iridoviruses grouped with phycodnaviruses-mimiviruses. The phycodna-mimivirus grouping was strongly supported by several derived shared characters, which seemed to rule out the previously suggested basal position of the mimivirus [Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, P., Ogata, H., La Scola, B., Suzan, M., Claverie, J.M. 2004. The 1.2-megabase genome sequence of Mimivirus. Science 306 (5700), 1344-1350]. These results indicate that the divergence of the major NCLDV families occurred at an early stage of evolution, prior to the divergence of the major eukaryotic lineages. It is shown that subsequent evolution of the NCLDV genomes involved lineage-specific expansion of paralogous gene families and acquisition of numerous genes via horizontal gene transfer from the eukaryotic hosts, other viruses, and bacteria (primarily, endosymbionts and parasites). Amongst the expansions, there are multiple families of predicted virus-specific signaling and regulatory domains. Most NCLDVs have also acquired large arrays of genes related to ubiquitin signaling, and the animal viruses in particular have independently evolved several defenses against apoptosis and immune response, including growth factors and potential inhibitors of cytokine signaling. The mimivirus displays an enormous array of genes of bacterial provenance, including a representative of a new class of predicted papainlike peptidases. It is further demonstrated that a significant number of genes found in NCLDVs also have homologs in bacteriophages, although a vertical relationship between the NCLDVs and a particular bacteriophage group could not be established. On the basis of these observations, two alternative scenarios for the origin of the NCLDVs and other groups of large DNA viruses of eukaryotes are considered. One of these scenarios posits an early assembly of an already large DNA virus precursor from which various large DNA viruses diverged through an ongoing process of displacement of the original genes by xenologous or non-orthologous genes from various sources. The second scenario posits convergent emergence, on multiple occasions, of large DNA viruses from small plasmid-like precursors through independent accretion of similar sets of genes due to strong selective pressures imposed by their life cycles and hosts. Published by Elsevier B.V. (L. Aravind). viruses: (1) their obligate intracellular parasitism; and (2) their virion architecture comprised of a genomic nucleic acid, typically of a single type (either RNA or DNA), packaged into a protein capsid, which in some cases is further associated with outer or inner lipid membranes . Beyond these general features, viruses show tremendous diversity in every respect, including genome size and organization, capsid architecture, mechanisms of propagation, and interactions with host cells. Viruses infect organisms from all three superkingdoms of life (bacteria, archaea, and eukaryotes) and replicate in all known 0168-1702/$ -see front matter. Published by Elsevier B.V.

sequence (Tanaka et al. 1995). The ORF contains 10 The product of the NS5B gene of Hepatitis C Virus genes of which those coding for structural proteins are (HCV) has been expressed in Escherichia Coli both as located at the 5 end, core, envelope 1 and envelope 2, a fusion protein with glutathione-S-transferase (GST) followed by the non-structural genes, NS2, NS3, NS4A, of molecular weight 91KDa and at high level as a single NS4B, NS5A, and NS5B (Grakoui et al. 1993 a). Proprotein of molecular weight 65KDa. The protein was cessing of the polyprotein has been analysed after exsequestered within inclusion bodies and a variety of pression of different genetically engineered recombiprocedures designed to minimize inclusion body fornant vectors, both procaryotic and eucaryotic, and after mation proved unsuccessful. The method finally in vitro translation of synthetic RNA. Structural genes

The organization of the eukaryotic genome into chromatin enables DNA to fit inside the nucleus while also regulating the access of proteins to the DNA to facilitate genomic functions such as transcription, replication and repair. The basic repeating unit of chromatin is the nucleosome, which includes 147 bp of DNA wrapped 1.65 times around an octamer of core histone proteins comprising two molecules each of H2A, H2B, H3 and H4 . Each nucleosome is a highly stable unit, being maintained by over 120 direct protein-DNA interactions and several hundred water mediated ones [1]. Accordingly, there is considerable interest in understanding how processive enzymes such as RNA polymerases manage to pass along the coding regions of our genes that are tightly packaged into arrays of nucleosomes. Here we present the current mechanistic understanding of this process and the evidence for profound changes in chromatin dynamics during aging. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly. j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g r m

The type 1 (microbial) rhodopsins are a diverse group of photochemically reactive proteins that display a broad yet patchy distribution among the three domains of life. Recent work indicates that this pattern is likely the result of lateral gene transfer (LGT) of rhodopsin genes between major lineages, and even across domain boundaries. Within the lineage in which the microbial rhodopsins were initially discovered, the haloarchaea, a similar patchy distribution is observed. In this initial study, we assess the roles of LGT and gene loss in the evolution of haloarchaeal rhodopsin ion pump genes, using phylogenetics and comparative genomics approaches.

Lipase genes originating from the Gram-negative bacteria Serrutiu marcescens and Pseudomonus urruginosa were cloned. S. marcescens lipase was overexpressed in Escherichia coli yielding inclusion bodies which were purified and finally refolded to give enzymatically active lipase. The lipase operon of P. aeruginosa consisting of genes 1ipA and lip14 was cloned behind the T7 410 promoter and overexpressed in a lipase-negative P. aeruginosa strain carrying a chromosomal insertion of the gene encoding T7 RNA polymerase. A 3D structural model was built for P. aeruginosu lipase using the coordinates of the Burkholderiu cepacia lipase structure which has recently been solved in its open conformation by X-ray crystallography.

Bacterial EBPs (enhancer-binding proteins) play crucial roles in regulating cellular responses to environmental changes, in part by providing efficient control over σ 54 -dependent gene transcription. The AAA+ (ATPase associated with various cellular activites) domain of the EBPs, when assembled into a ring, uses energy from ATP binding, hydrolysis and product release to remodel the σ 54 -RNAP (RNA polymerase) holoenzyme so that it can transition from closed to open form at promoter DNA. The assembly, and hence activity, of these ATPases are regulated by many different signal transduction mechanisms. Recent advances in solution scattering techniques, when combined with high-resolution structures and biochemical data, have enabled us to obtain mechanistic insights into the regulation and action of a subset of these σ 54 activators: those whose assembly into ring form is controlled by two-component signal transduction. We review (i) experimental considerations of applying the SAXS (small-angle X-ray scattering)/WAXS (wide-angle X-ray scattering) technique, (ii) distinct regulation mechanisms of the AAA+ domains of three EBPs by similar two-component signal transduction receiver domains, and (iii) major conformational changes and correlated σ 54binding activity of an isolated EBP AAA+ domain in the ATP hydrolysis cycle.

An RNA-dependent RNA polymerase (RdRp) activity was detergent-solubilized from the chloroplast membranes of Chinese cabbage leaves infected with turnip yellow mosaic virus (TYMV). The template-dependent, micrococcal nuclease-treated activity synthesized full-length minus strands from TYMV RNA and 3-fragments as short as a 28-nucleotide-long RNA comprising the amino acid acceptor stem of the 3-tRNA-like structure (TLS). Minus strands were shown to arise by de novo initiation with the insertion of GTP opposite the penultimate (C) residue of the 3-terminal -CCA. The TYMV RdRp activity was template specific in that poly(A) RNA was not copied, and alfalfa mosaic virus (AlMV) RNA, which does not contain a 3-TLS, was a very poor template. However, other viral RNAs with a 3-TLS and in vitro transcripts of tRNAs were copied to varying degrees. Fully modified tRNAs were either inactive or poorly active templates, and AlMV 3-RNA, even when provided with a 3-terminal -ACCA, was not copied detectably. A potential role of the acceptor stem pseudoknot as a promoter element was assessed with mutations that drastically altered the structure and sequence of the pseudoknot, revealing only a twofold effect in decreasing template activity. The data show that RNAs with both a tRNA-like conformation and a -CCA 3-terminus are potential templates for TYMV RdRp and suggest that promoter elements are not limited to the acceptor stem pseudoknot. ᭧ 1997 Academic Press

A collection of 247 true morels (Morchella spp.) primarily from the Mediterranean and Aegean Regions of Southern Turkey, were analyzed for species diversity using partial RNA polymerase I (RPB1) and nuclear ribosomal large subunit (LSU) rDNA gene sequences. Based on the result of this initial screen, 62 collections representing the full range of genetic diversity sampled were subjected to multigene phylogenetic species recognition based on genealogical concordance (GCPSR). The 62-taxon dataset consisted of partial sequences from three nuclear protein-coding genes, RNA polymerase I (RPB1), RNA polymerase II (RPB2), translation elongation factor (EF1-α), and partial LSU rDNA gene sequences. Phylogenetic analyses of the individual and combined datasets, using maximum parsimony (MP) and maximum likelihood (ML), yielded nearly fully resolved phylogenies that were highly concordant topologically. GCPSR analysis of the 62-taxon dataset resolved 15 putative phylogenetically distinct species. The early diverging Elata (black morels) and Esculenta Clades (yellow morels) were represented, respectively, by 13 and two species. Because a Latin binomial can be applied with confidence to only one of the 15 species (Morchella semilibera), species were identified by clade (Mel for Elata and Mes for Esculenta) followed by a unique Arabic number for each species within these two clades. Eight of the species within the Elata Clade appear to be novel, including all seven species within the Mel-20-to-31 subclade and its sister designated Mel-25. Results of the present study provide essential data for ensuring the sustainability of morel harvests through the formulation of sound conservation policies.