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.

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 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 The Bacteriophage T4 Transcription Activator MotA Interacts with the Far-C-Terminal Region of the σ70 Subunit of Escherichia coli RNA Polymerase

2002 ◽  
Vol 184 (14) ◽  
pp. 3957-3964 ◽  
Author(s):  
Suchira Pande ◽  
Anna Makela ◽  
Simon L. Dove ◽  
Bryce E. Nickels ◽  
Ann Hochschild ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
In Vitro Transcription ◽  
Terminal Region ◽  
Transcription Activator ◽  
E Coli ◽  
Terminal Amino ◽  
Two Hybrid

ABSTRACT Transcription from bacteriophage T4 middle promoters uses Escherichia coli RNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA. AsiA binds tightly within the C-terminal portion of the σ70 subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at −30, and also interacts with σ70. We show here that the N-terminal half of MotA (MotANTD), which is thought to include the activation domain, interacts with the C-terminal region of σ70 in an E. coli two-hybrid assay. Replacement of the C-terminal 17 residues of σ70 with comparable σ38 residues abolishes the interaction with MotANTD in this assay, as does the introduction of the amino acid substitution R608C. Furthermore, in vitro transcription experiments indicate that a polymerase reconstituted with a σ70 that lacks C-terminal amino acids 604 to 613 or 608 to 613 is defective for MotA-dependent activation. We also show that a proteolyzed fragment of MotA that contains the C-terminal half (MotACTD) binds DNA with a K D(app) that is similar to that of full-length MotA. Our results support a model for MotA-dependent activation in which protein-protein contact between DNA-bound MotA and the far-C-terminal region of σ70 helps to substitute functionally for an interaction between σ70 and a promoter −35 element.

scholarly journals Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter

2020 ◽  
pp. jbc.RA120.014553
Author(s):  
Zachary J Kartje ◽  
Helen I Janis ◽  
Shaoni Mukhopadhyay ◽  
Keith T Gagnon
Keyword(s):  
Rna Polymerase ◽  
Real Time ◽  
High Throughput Screening ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase ◽  
Rna Aptamer ◽  
Reaction Conditions ◽  
Fluorescent Reporter ◽  
Transcription In Vitro

Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, co-variation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.

scholarly journals Transcription in vitro of T3 DNA by Escherichia coli and T3 RNA Polymerases. Analysis of the Products in Cell-Free Protein-Synthesizing System

1972 ◽  
Vol 29 (3) ◽  
pp. 500-508 ◽  
Author(s):  
John J. Dunn ◽  
William T. Mcallister ◽  
Ekkehard K. F. Bautz
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerases ◽  
Free Protein ◽  
Transcription In Vitro

scholarly journals DksA Is Required for Growth Phase-Dependent Regulation, Growth Rate-Dependent Control, and Stringent Control of fis Expression in Escherichia coli

2006 ◽  
Vol 188 (16) ◽  
pp. 5775-5782 ◽  
Author(s):  
Prabhat Mallik ◽  
Brian J. Paul ◽  
Steven T. Rutherford ◽  
Richard L. Gourse ◽  
Robert Osuna
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Growth Phase ◽  
Stationary Growth ◽  
Rate Dependent ◽  
Transcription In Vitro ◽  
State Growth

ABSTRACT DksA is a critical transcription factor in Escherichia coli that binds to RNA polymerase and potentiates control of rRNA promoters and certain amino acid promoters. Given the kinetic similarities between rRNA promoters and the fis promoter (Pfis), we investigated the possibility that DksA might also control transcription from Pfis. We show that the absence of dksA extends transcription from Pfis well into the late logarithmic and stationary growth phases, demonstrating the importance of DksA for growth phase-dependent regulation of fis. We also show that transcription from Pfis increases with steady-state growth rate and that dksA is absolutely required for this regulation. In addition, both DksA and ppGpp are required for inhibition of Pfis promoter activity following amino acid starvation, and these factors act directly and synergistically to negatively control Pfis transcription in vitro. DksA decreases the half-life of the intrinsically short-lived fis promoter-RNA polymerase complex and increases its sensitivity to the concentration of CTP, the predominant initiating nucleotide triphosphate for this promoter. This work extends our understanding of the multiple factors controlling fis expression and demonstrates the generality of the DksA requirement for regulation of kinetically similar promoters.

scholarly journals Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ28 RNA Polymerase

2006 ◽  
Vol 188 (15) ◽  
pp. 5524-5531 ◽  
Author(s):  
Hilda Hiu Yin Yu ◽  
Elizabeth G. Di Russo ◽  
Megan A. Rounds ◽  
Ming Tan
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
Promoter Sequence ◽  
Promoter Element ◽  
Developmental Gene ◽  
Spacer Length ◽  
E Coli ◽  
Promoter Recognition ◽  
Developmental Gene Regulation

ABSTRACT σ28 RNA polymerase is an alternative RNA polymerase that has been postulated to have a role in developmental gene regulation in Chlamydia. Although a consensus bacterial σ28 promoter sequence has been proposed, it is based on a relatively small number of defined promoters, and the promoter structure has not been systematically analyzed. To evaluate the sequence of the σ28-dependent promoter, we performed a comprehensive mutational analysis of the Chlamydia trachomatis hctB promoter, testing the effect of point substitutions on promoter activity. We defined a −35 element recognized by chlamydial σ28 RNA polymerase that resembles the consensus −35 sequence. Within the −10 element, however, chlamydial σ28 RNA polymerase showed a striking preference for a CGA sequence at positions −12 to −10 rather than the longer consensus −10 sequence. We also observed a strong preference for this CGA sequence by Escherichia coli σ28 RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the −10 promoter element recognized by σ28 RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that σ28 RNA polymerase from both Chlamydia and E. coli transcribed a promoter with either an 11- or 12-nt spacer equally well. Altogether, we found very similar results for σ28 RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. The preferred σ28 promoter that we defined in the context of the hctB promoter is TAAAGwwy-n11/12-ryCGAwrn, where w is A or T, r is a purine, y is a pyrimidine, n is any nucleotide, and n11/12 is a spacer of 11 or 12 nt.

Vibrio harveyi RNA polymerase: purification and resolution from gyrase A

1992 ◽  
Vol 70 (8) ◽  
pp. 698-702 ◽  
Author(s):  
Elana Swartzman ◽  
Edward A. Meighen
Keyword(s):  
Rna Polymerase ◽  
Topoisomerase Ii ◽  
Vibrio Harveyi ◽  
In Vitro Transcription ◽  
E Coli ◽  
Gyrase A ◽  
A Subunit ◽  
Terminal Amino ◽  
Promoter Specificity

RNA polymerase was purified from Vibrio harveyi and found to contain polypeptides (β,β′, α, and σ) closely corresponding to those of the Escherichia coli enzyme. In vitro transcription studies using V. harveyi and E. coli RNA polymerase demonstrated that the purified V. harveyi RNA polymerase is functional and that the two enzymes have the same promoter specificity. Chromatography through a monoQ column was required to remove a 100-kilodalton protein that was present in large amounts and copurified with the RNA polymerase. N-terminal amino acid sequencing showed that the first 18 amino acids of the 100-kilodalton protein shares 78% sequence identity with the A subunit of gyrase or topoisomerase II. The abundance of the gyrase A protein is unprecedented and may be linked to bioluminescence.Key words: Vibrio harveyi, RNA polymerase, gyrase, bioluminescence.

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