scholarly journals Identification and Characterization of the dps Promoter of Mycobacterium smegmatis: Promoter Recognition by Stress-Specific Extracytoplasmic Function Sigma Factors σH and σF

2007 ◽  
Vol 189 (24) ◽  
pp. 8973-8981 ◽  
Author(s):  
Rakhi Pait Chowdhury ◽  
Surbhi Gupta ◽  
Dipankar Chatterji
Keyword(s):  
Rna Polymerase ◽  
Mycobacterium Smegmatis ◽  
Promoter Region ◽  
Sigma Factors ◽  
Galactosidase Activity ◽  
Putative Promoter ◽  
Activity Measurements ◽  
Promoter Recognition

ABSTRACT The survival of a bacterium with a depleted oxygen or nutrient supply is important for its long-term persistence inside the host under stressful conditions. We studied a gene, dps, from Mycobacterium smegmatis, encoding a protein, Dps (for DNA binding protein from starved cells), which is overexpressed under oxidative and nutritional stresses and provides bimodal protection to the bacterial DNA. Characterization of the dps promoter in vivo is therefore important. We cloned a 1-kb putative promoter region of the dps gene of M. smegmatis in an Escherichia coli-Mycobacterium shuttle vector, pSD5B, immediately upstream of the lacZ gene. Promoter activities were assayed in vivo both in solid medium and in liquid cultures by quantitative β-galactosidase activity measurements. To characterize the minimal promoter region, a 200-bp fragment from the whole 1-kb sequence was further cloned in the same vector, and in a similar way, β-galactosidase activity was quantitated. Primer extension analysis was performed to determine the +1 transcription start site of the gene. Point mutations were inserted in the putative promoter sequences in the −10 and −20 regions, and the promoter sequence was confirmed. The promoter was not recognized by purified M. smegmatis core RNA polymerase reconstituted with purified Mycobacterium tuberculosis σA or σB during multiple- and single-round in vitro transcription assays. Promoter-specific in vivo pull-down assays with an immobilized 1-kb DNA fragment containing the dps promoter established that extracellular function sigma factors were associated with this starvation-inducible promoter. Single-round transcription at the dps promoter further supported the idea that only core RNA polymerase reconstituted with σF or σH can generate proper transcripts.

scholarly journals Mutational Analysis of an Extracytoplasmic-Function Sigma Factor To Investigate Its Interactions with RNA Polymerase and DNA

2006 ◽  
Vol 188 (5) ◽  
pp. 1935-1942 ◽  
Author(s):  
Megan J. Wilson ◽  
Iain L. Lamont
Keyword(s):  
Rna Polymerase ◽  
Protein Function ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Alanine Scanning Mutagenesis ◽  
Sequence Alignments ◽  
Mutant Proteins ◽  
Promoter Recognition ◽  
Promoter Dna

ABSTRACT The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function.

scholarly journals Characterization of three cDNA species encoding plastid RNA polymerase sigma factors in Arabidopsis thaliana : evidence for the sigma factor heterogeneity in higher plant plastids

FEBS Letters ◽  
1997 ◽  
Vol 413 (2) ◽  
pp. 309-313 ◽  
Author(s):  
Kan Tanaka ◽  
Yuzuru Tozawa ◽  
Nobuyoshi Mochizuki ◽  
Kazuo Shinozaki ◽  
Akira Nagatani ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Higher Plant ◽  
Plastid Rna Polymerase

scholarly journals Sigma Factor F Does Not Prevent Rifampin Inhibition of RNA Polymerase or Cause Rifampin Tolerance in Mycobacterium tuberculosis

2010 ◽  
Vol 192 (20) ◽  
pp. 5472-5479 ◽  
Author(s):  
Ruben C. Hartkoorn ◽  
Claudia Sala ◽  
Sophie J. Magnet ◽  
Jeffrey M. Chen ◽  
Florence Pojer ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Potent Inhibitor ◽  
Sigma Factors ◽  
Antituberculosis Drugs ◽  
Axenic Cultures

ABSTRACT The tolerance of Mycobacterium tuberculosis to antituberculosis drugs is a major reason for the lengthy therapy needed to treat a tuberculosis infection. Rifampin is a potent inhibitor of RNA polymerase (RNAP) in vivo but has been shown to be less effective against stationary-phase bacteria. Sigma factor F is associated with bacteria entering stationary phase and has been proposed to impact rifampin activity. Here we investigate whether RNAP containing SigF is more resistant to rifampin inhibition in vitro and whether overexpression of sigF renders M. tuberculosis more tolerant to rifampin. Real-time and radiometric in vitro transcription assays revealed that rifampin equally inhibits transcription by RNAP containing sigma factors SigA and SigF, therefore ruling out the hypothesis that SigF may be responsible for increased resistance of the enzyme to rifampin in vitro. In addition, overexpression or deletion of sigF did not alter rifampin susceptibility in axenic cultures of M. tuberculosis, indicating that SigF does not affect rifampin tolerance in vivo.

scholarly journals ςBldN, an Extracytoplasmic Function RNA Polymerase Sigma Factor Required for Aerial Mycelium Formation in Streptomyces coelicolor A3(2)

2000 ◽  
Vol 182 (16) ◽  
pp. 4606-4616 ◽  
Author(s):  
Maureen J. Bibb ◽  
Virginie Molle ◽  
Mark J. Buttner
Keyword(s):  
Rna Polymerase ◽  
Sigma Factor ◽  
Streptomyces Coelicolor ◽  
Aerial Mycelium ◽  
Sigma Factors ◽  
Colony Development ◽  
Null Mutant ◽  
Wild Type

ABSTRACT Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci. whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N′-nitro-N-nitrosoguanidine)-inducedwhi strains (N. J. Ryding et al., J. Bacteriol. 181:5419–5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed thatwhiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed whiN null mutant failed to form aerial mycelium (the “bald” phenotype) and, as a consequence, whiN was renamed bldN. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S. coelicolor bld mutants. Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficientbld strains. bldN was transcribed from a single promoter, bldNp. bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC,bldF, bldK, or bldJ or onbldN itself. Transcription from the p1 promoter of the response-regulator gene bldM depended onbldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for ςBldN holoenzyme in vitro.

scholarly journals In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading

2003 ◽  
Vol 33 (4) ◽  
pp. 469-475 ◽  
Author(s):  
Julian C. Knight ◽  
Brendan J. Keating ◽  
Kirk A. Rockett ◽  
Dominic P. Kwiatkowski
Keyword(s):  
Rna Polymerase ◽  
Allele Specific ◽  
In Vivo Characterization

scholarly journals RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.

1986 ◽  
Vol 6 (11) ◽  
pp. 3984-3989 ◽  
Author(s):  
D S Gilmour ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Promoter Region ◽  
High Energy ◽  
Heat Shock Gene ◽  
Cross Linking ◽  
Hsp70 Gene

By using a protein-DNA cross-linking method (D. S. Gilmour and J. T. Lis, Mol. Cell. Biol. 5:2009-2018, 1985), we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.

scholarly journals Genomic organization and in vivo characterization of proteolytic activity of FtsH of Mycobacterium smegmatis SN2

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2629-2639 ◽  
Author(s):  
Gopalakrishnapillai Anilkumar ◽  
Ramanujam Srinivasan ◽  
Parthasarathi Ajitkumar
Keyword(s):  
Mycobacterium Smegmatis ◽  
Genomic Dna ◽  
Genomic Organization ◽  
Growth Arrest ◽  
Upstream Region ◽  
E Coli ◽  
Dna Libraries ◽  
In Vivo Characterization

The ftsH gene of Mycobacterium smegmatis SN2 (MsftsH) was cloned from two independent partial genomic DNA libraries and characterized, along with the identification of ephA and folE as the neighbouring upstream and downstream genes respectively. The genomic organization of the MsftsH locus was found to be identical to that of the Mycobacterium tuberculosis ftsH gene (MtftsH) and similar to that of other bacterial genera, but with divergence in the upstream region. The MsftsH gene is 2·3 kb in size and encodes the AAA (ATPases Associated with diverse cellular Activities) family Zn2+-metalloprotease FtsH (MsFtsH) of 85 kDa molecular mass. This was demonstrated from the expression of the full-length recombinant gene in Escherichia coli JM109 cells and from the identification of native MsFtsH in M. smegmatis SN2 cell lysates by Western blotting with anti-MtFtsH and anti-EcFtsH antibodies respectively. The recombinant and the native MsFtsH proteins were found localized to the membrane of E. coli and M. smegmatis cells respectively. Expression of MsFtsH protein in E. coli was toxic and resulted in growth arrest and filamentation of cells. The MsftsH gene did not complement lethality of a ΔftsH3 : : kan mutation in E. coli, but when expressed in E. coli cells, it efficiently degraded conventional FtsH substrates, namely σ 32 protein and the protein translocase subunit SecY, of E. coli cells.

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