scholarly journals Two Extracytoplasmic Function Sigma Subunits, ςE and ςFecI, of Escherichia coli: Promoter Selectivity and Intracellular Levels

2000 ◽  
Vol 182 (4) ◽  
pp. 1181-1184 ◽  
Author(s):  
Hiroto Maeda ◽  
Miki Jishage ◽  
Tasuku Nomura ◽  
Nobuyuki Fujita ◽  
Akira Ishihama
Keyword(s):  
Escherichia Coli ◽  
In Vitro Transcription ◽  
High Rate ◽  
State Culture ◽  
E Coli ◽  
Transcription System ◽  
Iron Sequestration ◽  
Promoter Recognition ◽  
High Concentrations

ABSTRACT The promoter selectivity of two extracytoplasmic function (ECF) subfamily ς subunits, ςE (ς24) and ςFecI (ς18), of Escherichia coli RNA polymerase was analyzed by using an in vitro transcription system and various promoters. The EςEholoenzyme recognized only the known cognate promoters,rpoEP2, rpoHP3, and degP, and the EςFecI recognized only one known cognate promoter,fecA. The strict promoter recognition properties of ςE and ςFecI are similar to those of other minor ς subunits. Transcription by EςE and EςFecI was enhanced by high concentrations of glutamate, as in the case of other minor ς subunits. The optimum temperature for transcription by EςFecI was low, around 25°C, apparently in agreement with the high rate of iron sequestration byE. coli at low temperatures. By quantitative Western blot analysis, the intracellular levels of ςE and ςFecI in the uninduced steady-state culture of E. coli W3110 (type A) were determined to be 0.7 to 2.0 and 0.1 to 0.2 fmol per μg of total proteins (or 3 to 9 and 0.4 to 0.9 molecules per cell), respectively, and less than 1% of the level of the major ς70 subunit.

scholarly journals Hybrid Bordetella pertussis-Escherichia coli RNA Polymerases: Selectivity of Promoter Activation

1998 ◽  
Vol 180 (6) ◽  
pp. 1567-1569 ◽  
Author(s):  
Pierre Steffen ◽  
Agnes Ullmann
Keyword(s):  
Escherichia Coli ◽  
Bordetella Pertussis ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Α Subunit ◽  
E Coli ◽  
Transcription System ◽  
Catabolite Gene Activator Protein ◽  
Transcription Activators

ABSTRACT We constructed hybrid Bordetella pertussis-Escherichia coli RNA polymerases and compared productive interactions between transcription activators and cognate RNA polymerase subunits in an in vitro transcription system. Virulence-associated genes of B. pertussis, in the presence of their activator BvgA, are transcribed by all variants of hybrid RNA polymerases, whereas transcription at the E. coli lacpromoter regulated by the cyclic AMP-catabolite gene activator protein has an absolute requirement for the E. coli α subunit. This suggests that activator contact sites involve a high degree of selectivity.

scholarly journals RNA Polymerases from Bacillus subtilisand Escherichia coli Differ in Recognition of Regulatory Signals In Vitro

2000 ◽  
Vol 182 (21) ◽  
pp. 6027-6035 ◽  
Author(s):  
Irina Artsimovitch ◽  
Vladimir Svetlov ◽  
Larry Anthony ◽  
Richard R. Burgess ◽  
Robert Landick
Keyword(s):  
Escherichia Coli ◽  
Bacterial Species ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Bacterial Cells ◽  
Signal Recognition ◽  
E Coli ◽  
Promoter Recognition ◽  
Species Specific

ABSTRACT Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals. Some of these signals are conserved throughout evolution, whereas others are species specific. In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species, Escherichia coli and Bacillus subtilis, using a panel of in vitro transcription assays. We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals. Both enzymes responded similarly to other pause and termination signals and to the general E. coli elongation factors NusA and GreA. We also demonstrate that, although promoter recognition depends largely on the ς subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme. We hypothesize that differences in signal recognition are due to the changes in contacts made between the β and β′ subunits and the downstream DNA duplex.

scholarly journals A Positive cis-Acting DNA Element Is Required for High-Level Transcription in Chlamydia

2000 ◽  
Vol 182 (18) ◽  
pp. 5167-5171 ◽  
Author(s):  
Chris S. Schaumburg ◽  
Ming Tan
Keyword(s):  
Escherichia Coli ◽  
Chlamydia Trachomatis ◽  
Rna Polymerase ◽  
Promoter Region ◽  
In Vitro Transcription ◽  
Transcription Assay ◽  
Cis Acting ◽  
E Coli ◽  
High Level

ABSTRACT The spacer A/T region is a positive cis-acting DNA element that was identified in the Chlamydia trachomatisrRNA promoter region. We have now demonstrated that similar sequences in other chlamydial promoters are important for transcription. Substitution of candidate spacer A/T regions in four chlamydial promoters decreased transcription by partially purified C. trachomatis RNA polymerase in an in vitro transcription assay. Addition of a spacer A/T region to the dnaK promoter, which does not contain an identifiable spacer A/T region, increased transcription 16-fold. Transcription of Escherichia colipromoters by C. trachomatis RNA polymerase also appeared to be dependent on the spacer A/T region. However, the effect of the spacer A/T region on transcription by E. coli RNA polymerase was small. In summary, the spacer A/T region is a novel DNA element that is required for high-level transcription of many promoters by chlamydial RNA polymerase.

scholarly journals Mutational Analysis of the Chlamydia trachomatis rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro

1998 ◽  
Vol 180 (9) ◽  
pp. 2359-2366 ◽  
Author(s):  
Ming Tan ◽  
Tamas Gaal ◽  
Richard L. Gourse ◽  
Joanne N. Engel
Keyword(s):  
Escherichia Coli ◽  
Chlamydia Trachomatis ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Rich Region ◽  
E Coli ◽  
Transcription In Vitro

ABSTRACT We have characterized the Chlamydia trachomatisribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purifiedC. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous toEscherichia coli promoters recognized by E-ς70. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I

1985 ◽  
Vol 5 (6) ◽  
pp. 1358-1369
Author(s):  
R M Learned ◽  
S Cordes ◽  
R Tjian
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Rna Polymerase I ◽  
Control Element ◽  
In Vitro Synthesis ◽  
Transcription System ◽  
Activation Of Transcription ◽  
Promoter Recognition ◽  
Polymerase I

A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have shown that the addition of SL1 can reprogram the otherwise nonpermissive mouse transcription system to recognize and initiate accurate RNA synthesis from human rDNA. Antibodies raised against SL1 bind preferentially to a protein localized in the nucleolus of primate cells and specifically inhibit in vitro transcription initiating from the human rRNA promoter. By contrast, anti-SL1 does not react with the nucleolus of rodent cells and has no effect on the in vitro synthesis of mouse rRNA by a transcription system derived from mouse cells. These findings suggest that SL1 is a selectivity factor present in the nucleolus that imparts promoter recognition to RNA polymerase I and that can discriminate between rRNA promoters from different species.

scholarly journals The Chromosomal Arsenic Resistance Genes of Thiobacillus ferrooxidans Have an Unusual Arrangement and Confer Increased Arsenic and Antimony Resistance to Escherichia coli

2000 ◽  
Vol 66 (5) ◽  
pp. 1826-1833 ◽  
Author(s):  
Bronwyn G. Butcher ◽  
Shelly M. Deane ◽  
Douglas E. Rawlings
Keyword(s):  
Escherichia Coli ◽  
Resistance Genes ◽  
Thiobacillus Ferrooxidans ◽  
In Vitro Transcription ◽  
Translation System ◽  
Gram Positive ◽  
Arsenic Resistance ◽  
E Coli ◽  
Gram Staining

ABSTRACT The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium Thiobacillus ferrooxidans were cloned and sequenced. Homologues of four arsenic resistance genes, arsB, arsC,arsH, and a putative arsR gene, were identified. The T. ferrooxidans arsB (arsenite export) andarsC (arsenate reductase) gene products were functional when they were cloned in an Escherichia coli ars deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the ars genes originated from an obligately acidophilic bacterium, they were functional in E. coli. Although T. ferrooxidansis gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functionaltrxA (thioredoxin) gene was required for ArsC-mediated arsenate resistance in E. coli; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although arsH was expressed in an E. coli-derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in E. coli. The T. ferrooxidans ars genes were arranged in an unusual manner, and the putative arsR andarsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated.

scholarly journals Escherichia coli Promoters with UP Elements of Different Strengths: Modular Structure of Bacterial Promoters

1998 ◽  
Vol 180 (20) ◽  
pp. 5375-5383 ◽  
Author(s):  
Wilma Ross ◽  
Sarah E. Aiyar ◽  
Julia Salomon ◽  
Richard L. Gourse
Keyword(s):  
Escherichia Coli ◽  
Core Promoter ◽  
Consensus Sequence ◽  
Α Subunit ◽  
Core Promoters ◽  
E Coli ◽  
Promoter Recognition ◽  
Transcription In Vitro ◽  
Up Elements

ABSTRACT The α subunit of Escherichia coli RNA polymerase (RNAP) participates in promoter recognition through specific interactions with UP element DNA, a region upstream of the recognition hexamers for the ς subunit (the −10 and −35 hexamers). UP elements have been described in only a small number of promoters, including the rRNA promoter rrnB P1, where the sequence has a very large (30- to 70-fold) effect on promoter activity. Here, we analyzed the effects of upstream sequences from several additional E. coli promoters (rrnD P1, rrnB P2, λp R, lac, merT, and RNA II). The relative effects of different upstream sequences were compared in the context of their own core promoters or as hybrids to thelac core promoter. Different upstream sequences had different effects, increasing transcription from 1.5- to ∼90-fold, and several had the properties of UP elements: they increased transcription in vitro in the absence of accessory protein factors, and transcription stimulation required the C-terminal domain of the RNAP α subunit. The effects of the upstream sequences correlated generally with their degree of similarity to an UP element consensus sequence derived previously. Protection of upstream sequences by RNAP in footprinting experiments occurred in all cases and was thus not a reliable indicator of UP element strength. These data support a modular view of bacterial promoters in which activity reflects the composite effects of RNAP interactions with appropriately spaced recognition elements (−10, −35, and UP elements), each of which contributes to activity depending on its similarity to the consensus.

scholarly journals Nitrogen Regulation of the codBA (Cytosine Deaminase) Operon from Escherichia coli by the Nitrogen Assimilation Control Protein, NAC

2003 ◽  
Vol 185 (9) ◽  
pp. 2920-2926 ◽  
Author(s):  
Wilson B. Muse ◽  
Christopher J. Rosario ◽  
Robert A. Bender
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Assimilation ◽  
Cytosine Deaminase ◽  
In Vitro Transcription ◽  
Dependent Manner ◽  
E Coli ◽  
Different Response ◽  
Control Protein

ABSTRACT Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. β-Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position −59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions −120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions −83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.

scholarly journals Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I.

1985 ◽  
Vol 5 (6) ◽  
pp. 1358-1369 ◽  
Author(s):  
R M Learned ◽  
S Cordes ◽  
R Tjian
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Rna Polymerase I ◽  
Control Element ◽  
In Vitro Synthesis ◽  
Transcription System ◽  
Activation Of Transcription ◽  
Promoter Recognition ◽  
Polymerase I

A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have shown that the addition of SL1 can reprogram the otherwise nonpermissive mouse transcription system to recognize and initiate accurate RNA synthesis from human rDNA. Antibodies raised against SL1 bind preferentially to a protein localized in the nucleolus of primate cells and specifically inhibit in vitro transcription initiating from the human rRNA promoter. By contrast, anti-SL1 does not react with the nucleolus of rodent cells and has no effect on the in vitro synthesis of mouse rRNA by a transcription system derived from mouse cells. These findings suggest that SL1 is a selectivity factor present in the nucleolus that imparts promoter recognition to RNA polymerase I and that can discriminate between rRNA promoters from different species.

Reduction of an Escherichia coli K12 Population by Bdellovibrio bacteriovorus Under Various In Vitro Conditions of Parasite:Host Ratio, Temperature, or pH

1992 ◽  
Vol 55 (11) ◽  
pp. 859-861 ◽  
Author(s):  
LEEANNE JACKSON ◽  
RICHARD C. WHITING
Keyword(s):  
Escherichia Coli ◽  
Bacterial Population ◽  
Bdellovibrio Bacteriovorus ◽  
Gram Negative ◽  
E Coli ◽  
Log Reduction ◽  
Extrinsic Parameters ◽  
High Concentrations ◽  
Intrinsic And Extrinsic Parameters

Intrinsic and extrinsic parameters of food products, as well as bacterial population, were evaluated for their effects on the ability of Bdellovibrio bacteriovorus, a bacterium parasitic upon gram-negative bacteria, to reduce an Escherichia coli population. High concentrations of both parasite and host were the most effective for reducing a specified E. coli population. B. bacteriovorus was able to reduce the E. coli count by 90% (1 log) in < 1 h at ratios of 5:1, 10:1, and 30:1 (parasite:host). Temperatures between 20 and 30°C were more conducive to bdellovibrio attack than temperatures less than 20°C. E. coli populations were reduced by more than 7-log values after 7 h of incubation at 30°C with parasite:host ratios of 2:1, 5:1, and 10:1. Greater than a 5-log reduction in the E. coli population was observed at the ratio of 30:1. B. bacteriovorus reduced the E. coli population by 1 log in approximately 24 min and 20 min at pH 7.2 and 6.8, respectively. At pH values <6.8, the activity of B. bacteriovorus was diminished. These results define some of the conditions where the application of B. bacteriovorus may aid in the reduction/elimination of some gram-negative pathogens and spoilage flora that may be present in foods.

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