Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

By using a PCR approach, the Bradyrhizobium japonicum sigA gene, which encodes the primary RNA polymerase sigma factor, sigma80, was cloned and its nucleotide sequence was established. The deduced protein is highly homologous to the SigA protein of Rhizobium meliloti (72% amino acid sequence identity) but less so to RpoD of Escherichia coli (51% identity). Well conserved is the C-terminal end of the protein, which is probably involved in promoter recognition and binding of the RNA polymerase core enzyme. A remarkable feature of the primary sequence is an alanine- and proline-rich segment of 24 amino acids between conserved regions 1 and 2, which might function as an interdomain linker. We purified the B. japonicum RNA polymerase holoenzyme. One of the subunits had an apparent molecular mass of 90 kDa and corresponded to the sigA gene product, as judged by N-terminal amino acid sequencing. The purified RNA polymerase was used in an in vitro transcription system to determine the transcription start sites of the rrn and groESL4 operons. They were identical to those previously identified in vivo. The rrn promoter was cloned upstream of a rho-independent terminator, yielding a transcript of about 240 bases. This served as a suitable template to analyze promoter activity. Then mutant derivatives of the rrn promoter were constructed and tested in in vitro transcription experiments. Several base pairs essential for promoter activity were thus identified. The results suggest that the well-characterized -35/-10 promoter class is predominantly used in B. japonicum for the expression of "housekeeping" genes.
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PMID:Dissection of the transcription machinery for housekeeping genes of Bradyrhizobium japonicum. 899 Feb 87

Integration host factor (IHF) can activate transcription from the early promoter (Pe) of bacteriophage Mu both directly and indirectly. Indirect activation occurs through alleviation of H-NS-mediated repression of the Pe promoter (P. Van Ulsen, M. Hillebrand, L. Zulianello, P. Van de Putte, and N. Goosen, Mol. Microbiol. 21:567-578, 1996). The direct activation involves the C-terminal domain of the alpha subunit (alphaCTD) of RNA polymerase. We investigated which residues in the alphaCTD are important for IHF-mediated activation of the Pe promoter. Initial in vivo screening, using a set of substitution mutants derived from an alanine scan (T. Gaal, W. Ross, E. E. Blatter, T. Tang, X. Jia, V. V. Krishnan, N. Assa-Munt, R. Ebright, and R. L. Gourse, Genes Dev. 10:16-26, 1996; H. Tang, K. Severinov, A. Goldfarb, D. Fenyo, B. Chait, and R. H. Ebright, Genes Dev. 8:3058-3067, 1994), indicated that the residues, which are required for transcription activation by the UP element of the rrnB P1 promoter (T. Gaal, W. Ross, E. E. Blatter, T. Tang, X. Jia, V. V. Krishnan, N. Assa-Munt, R. Ebright, and R. L. Gourse, Genes Dev. 10:16-26, 1996), are also important for Pe expression in the presence of IHF. Two of the RNA polymerase mutants, alphaR265A and alphaG296A, that affected Pe expression most in vivo were subsequently tested in in vitro transcription experiments. Mutant RNA polymerase with alphaR265A showed no IHF-mediated activation and a severely reduced basal level of transcription from the Pe promoter. Mutant RNA polymerase with alphaG296A resulted in a slightly reduced transcription from the Pe promoter in the absence of IHF but could still be activated by IHF. These results indicate that interaction of the alphaCTD with DNA is involved not only in the IHF-mediated activation of Pe transcription but also in maintaining the basal level of transcription from this promoter. Mutational analysis of the upstream region of the Pe promoter identified a sequence, positioned from -39 to -51 with respect to the transcription start site, that is important for basal Pe expression, presumably through binding of the alphaCTD. The role of the alphaCTD in IHF-mediated stimulation of transcription from the Pe promoter is discussed.
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PMID:Function of the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase in basal expression and integration host factor-mediated activation of the early promoter of bacteriophage Mu. 899 Mar 7

The nucleocapsid protein (NP) of Sendai virus encapsidates the genome RNA, forming a helical nucleocapsid which is the template for RNA synthesis by the viral RNA polymerase. The NP protein is thought to have both structural and functional roles, since it is an essential component of the NP0-P (P, phosphoprotein), NP-NP, nucleocapsid-polymerase, and RNA-NP complexes required during viral RNA replication. To identify domains in the NP protein, mutants were constructed by using clustered charge-to-alanine mutagenesis in a highly charged region from amino acids 107 to 129. Each of the mutants supported RNA encapsidation in vitro. The product nucleocapsids formed with three mutants, NP114, NP121, and NP126, however, did not serve as templates for further amplification in vivo, while NP107, NP108, and NP111 were nearly like wild-type NP in vivo. This template defect in the NP mutants from amino acids 114 to 129 was not due to a lack of NP0-P, NP-NP, or nucleocapsid-polymerase complex formation, since these interactions were normal in these mutants. We propose that amino acids 114 to 129 of the NP protein are required for the nucleocapsid to function as a template in viral genome replication.
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PMID:An amino-terminal domain of the Sendai virus nucleocapsid protein is required for template function in viral RNA synthesis. 899 8

In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.
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PMID:Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2). 902 6

A number of Escherichia coli clones were isolated from a Lactobacillus delbrueckii subsp. lactis gene library capable of hydrolysing the chromogenic substrate Gly-Ala-beta-naphthylamide (Gly-Ala-beta NA). Some of the recombinant plasmids carried by these clones have been shown to encode the cysteine aminopeptidase gene pepC. Nucleotide sequence analyses of the plasmid inserts of the remaining clones resulted in the identification of two adjacent ORFs encoding proteins exhibiting a high degree of similarity between themselves (72.6%) and with PepC. One gene, designated pepG, was overexpressed in E. coli and the crude extracts obtained were shown to be peptidolytically active both against chromogenic substrates and peptides, and in a Salmonella typhimurium growth test. PepC and PepG activities were compared using chromogenic beta NA and p-nitroanilide substrates and leucine or proline-containing peptides were applied in growth experiments of recombinant Sal. typhimurium. The results indicate that the enzymes, although structurally related, have different substrate preferences. No enzyme activity could be ascribed to the second ORF (orfW), despite the production of a visible protein using a T7 RNA polymerase system. Primer extension analysis, using mRNA isolated from Lb. delbrueckii subsp. lactis DSM7290 did establish that orfW was transcribed.
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PMID:Lactobacillus delbrueckii subsp. lactis DSM7290 pepG gene encodes a novel cysteine aminopeptidase. 904 29

The expression of virulence factor genes in Bordetella pertussis is mediated by the BvgA-BvgS two-component signal transduction system. The response regulator, BvgA, acts directly as a transcriptional activator at the loci encoding pertussis toxin (ptx) and filamentous hemagglutinin (fha). Previous studies have demonstrated that these two loci are differentially regulated by BvgA. As an initial step in gaining insight into the mechanism underlying this differential regulation, we initiated DNA binding and in vitro transcription analyses to examine the activities of BvgA and RNA polymerase (RNAP) purified from both B. pertussis and Escherichia coli at the fha promoter. We discovered that unphosphorylated BvgA binds to a single region (-100 to -70, relative to the start of transcription), whereas phosphorylated BvgA binds both this region and another, farther downstream, that extends to the -35 nucleotide. In the absence of BvgA, RNAP binds a region farther upstream than expected (-104 to -35). However, occupation of both sites by BvgA phosphate repositions RNAP to the site used in vivo. The binding of BvgA phosphate to the downstream site correlates with in vitro transcriptional activity at the fha promoter. As the DNA binding and transcription activities of the E. coli-derived RNAP are similar to those observed for the B. pertussis enzyme, we employed several mutant E. coli proteins in in vitro transcription analyses. We observed that polymerases carrying either a deletion of the C-terminal domain of the alpha subunit or substitution of alanine at either of two critical residues within this domain were severely impaired in the ability to mediate BvgA-activated transcription at fha.
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PMID:Nature of DNA binding and RNA polymerase interaction of the Bordetella pertussis BvgA transcriptional activator at the fha promoter. 904 38

Bacteriophage T7 RNA polymerase is a single-subunit enzyme which has a C-terminal amino acid sequence of Phe-Ala-Phe-Ala883 (FAFA883). Closely related hydrophobic sequences are present at the C termini of seven other single-subunit RNA polymerases, including the mitochondrial RNA polymerase. Mutations at any of the four C-terminal residues depress initiation rates of T7 RNA polymerase from 50 to 95%, accompanied by large increases in the K(m) values for the initiating nucleotide, GTP, as well as the K(m)'s for promoter DNA. The dramatic drops in initiation rates shown by the mutant enzymes remain after correcting for any alteration in saturation of the enzyme by the initiating nucleotide or the promoter DNA resulting from the changes in K(m). In contrast, the high processivity of the enzyme is not altered by mutations in the last four residues. However, the propensity for the enzyme to add an untemplated nucleotide at the 3'-ends of transcripts is abolished by the A880AFA883 mutation. The C-terminal FAFA sequence or foot appears to interact both with the initiating NTP and with the most downstream nucleotides of the promoter, possibly through hydrophobic interactions with the minor groove, in the region where free radical footprinting of the polymerase-promoter DNA complex suggests that the enzyme binds across the minor groove.
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PMID:Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase. 906 20

Among various group I sigma factors, two amino acids, Val55 and Ala59 are the conserved amino acids in the 1.1 hydrophobic subdomain. These two sites have been mutated to generate variants designated as [Gly55]sigma70 and [Gly59]sigma70, where glycine replaces valine and alanine, respectively. The function of these sigma mutants is reported here. The molecular mass of these proteins determined on denaturing gels was 70 kDa, which is the expected calculated molecular mass; wild-type sigma70 has an apparent molecular mass of 87 kDa. However, [Gly434]sigma70, which contains a mutation at the DNA-binding rpoD box region, also migrates as a 70-kDa protein on SDS/PAGE. Circular dichroism spectral analysis indicated that both [Gly55]sigma70 and [Gly59]sigma70 have reduced helicity (20%) compared to wild-type sigma70 (50%). Binding of sigma factors with the hydrophobic, surface active probe 1-anilinonapthalene-8-sulphonate, has shown that more hydrophobic surfaces are available/exposed in [Gly55]sigma70, [Gly59]sigma70 as well as in [Gly434]sigma70 in comparison to wild-type sigma70. Time-resolved emission spectroscopic studies have suggested transient binding between these mutants and DNA. The different holoenzyme RNA polymerases generated upon reconstituting these mutants independently with core RNA polymerase (alpha2beta beta') have shown reduced transcriptional activity in comparison to the enzyme containing wild-type sigma factor. However, another mutation (Val-->Gly) in the hydrophobic subdomain 1.2 at position 83, which is designated as [Gly83]sigma70, has similar properties as the wild-type with respect to its mobility on denaturing gels, circular dichroism profile, and transcriptional activity when reconstituted with core RNA polymerase. It appears that the 1.1 subdomain in sigma70 may interact hydrophobically with the 2.3/2.4 DNA-binding region.
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PMID:Mutations in the 1.1 subdomain of Escherichia coli sigma factor sigma70 and disruption of its overall structure. 911 31

Members of the Bunyaviridae family mature by a budding process in the Golgi complex. The site of maturation is thought to be largely determined by the accumulation of the two spike glycoproteins, G1 and G2, in this organelle. Here we show that the signal for localizing the Uukuniemi virus (a phlebovirus) spike protein complex to the Golgi complex resides in the cytoplasmic tail of G1. We constructed chimeric proteins in which the ectodomain, transmembrane domain (TMD), and cytoplasmic tail (CT) of Uukuniemi virus G1 were exchanged with the corresponding domains of either vesicular stomatitis virus G protein (VSV G), chicken lysozyme, or CD4, all proteins readily transported to the plasma membrane. The chimeras were expressed in HeLa or BHK-21 cells by using either the T7 RNA polymerase-driven vaccinia virus system or the Semliki Forest virus system. The fate of the chimeric proteins was monitored by indirect immunofluorescence, and their localizations were compared by double labeling with markers specific for the Golgi complex. The results showed that the ectodomain and TMD (including the 10 flanking residues on either side of the membrane) of G1 played no apparent role in targeting chimeric proteins to the Golgi complex. Instead, all chimeras containing the CT of G1 were efficiently targeted to the Golgi complex and colocalized with mannosidase II, a Golgi-specific enzyme. Conversely, replacing the CT of G1 with that from VSV G resulted in the efficient transport of the chimeric protein to the cell surface. Progressive deletions of the G1 tail suggested that the Golgi retention signal maps to a region encompassing approximately residues 10 to 50, counting from the proposed border between the TMD and the tail. Both G1 and G2 were found to be acylated, as shown by incorporation of [3H]palmitate into the viral proteins. By mutational analyses of CD4-G1 chimeras, the sites for palmitylation were mapped to two closely spaced cysteine residues in the G1 tail. Changing either or both of these cysteines to alanine had no effect on the targeting of the chimeric protein to the Golgi complex.
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PMID:A retention signal necessary and sufficient for Golgi localization maps to the cytoplasmic tail of a Bunyaviridae (Uukuniemi virus) membrane glycoprotein. 915 65

Leukemia in the soft-shell clam, Mya arenaria, is characterized by tumor cells which are detected initially in the hemolymph. This disease is much more common in clams inhabiting polluted waters, suggesting an environmental component to its pathogenesis. In this study, leukemia cells were identified using a murine monoclonal antibody, 1E10, which recognizes a leukemia-specific protein expressed by tumor cells. Mutant p53 protein was detected using a murine monoclonal antibody (PAb 240) which reacts with mutant p53. Using immunofluorescence, the reactivity of clam cells to the 1E10 antibody was evaluated along with mutant p53 protein reactivity. Reverse transcriptase-polymerase chain reactions followed by sequence analyses were utilized to examine clams with hemocytes reacting with the p53 antibody for possible p53 gene mutations. Mutant p53 protein was expressed by tumor cells from five animals with advanced disease (in which greater than 90% of cells reacted with 1E10). A C-->G transversion was detected at the end of exon 6 from two of the five animals that reacted with both the mutant p53 antibody and 1E10. This substitution changes the amino acid of this codon from proline to alanine. Overall, our results suggest that environmentally induced alterations in p53 can contribute to the pathogenesis of leukemia in soft-shell clams inhabiting polluted water and/or sediment.
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PMID:Detection of mutant p53 in clam leukemia cells. 916 98


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