In vitro transcription of three different ribosomal RNA cistrons of E. coli; heterogeneity of control regions

1977 ◽  
Vol 152 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Ben A. Oostra ◽  
Albert J. J. van Ooyen ◽  
Max Gruber
Keyword(s):  
Ribosomal Rna ◽  
In Vitro Transcription ◽  
E Coli ◽  
Control Regions

In vitro transcription of the ribosomal RNA genes of E. coli DNA

1977 ◽  
Vol 151 (3) ◽  
pp. 305-312 ◽  
Author(s):  
János Sümegi ◽  
Andor Udvardy ◽  
Pál Venetianer
Keyword(s):  
Ribosomal Rna ◽  
In Vitro Transcription ◽  
Ribosomal Rna Genes ◽  
E Coli ◽  
Rna Genes

Cloning the gene for the heat shock response positiwe regulator (sigma 32 homolog) from Pseudomonas aeruginosa

1995 ◽  
Vol 41 (1) ◽  
pp. 75-87 ◽  
Author(s):  
Zerlina M. Naczynski ◽  
Andrew M. Kropinski ◽  
Chris Mueller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heat Shock ◽  
Sigma Factor ◽  
Oligonucleotide Probe ◽  
In Vitro Transcription ◽  
Translation System ◽  
Amino Acid Homology ◽  
E Coli ◽  
Transcriptional Start Sites

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040–1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (σ32) of E. coli. The plasmid containing the rpoH gene complemented the function of σ32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription–translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 °C) conditions. Two promoter sites were identified having sequence homology to the E. coli σ70 and σ24 consensus sequences.Key words: heat shock, Pseudomonas aeruginosa, sigma factor, transcription, oligonucleotide probe.

scholarly journals Structural basis for transcription inhibition by E. coli SspA

2020 ◽  
Vol 48 (17) ◽  
pp. 9931-9942 ◽  
Author(s):  
Fulin Wang ◽  
Jing Shi ◽  
Dingwei He ◽  
Bei Tong ◽  
Chao Zhang ◽  
...  
Keyword(s):  
Structural Information ◽  
Protein A ◽  
Acid Tolerance ◽  
In Vitro Transcription ◽  
Nucleotide Metabolism ◽  
Structural Basis ◽  
E Coli ◽  
Promoter Escape ◽  
Zinc Binding Domain

Abstract Stringent starvation protein A (SspA) is an RNA polymerase (RNAP)-associated protein involved in nucleotide metabolism, acid tolerance and virulence of bacteria. Despite extensive biochemical and genetic analyses, the precise regulatory role of SspA in transcription is still unknown, in part, because of a lack of structural information for bacterial RNAP in complex with SspA. Here, we report a 3.68 Å cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA. Unexpectedly, the structure reveals that SspA binds to the E. coli σ70-RNAP holoenzyme as a homodimer, interacting with σ70 region 4 and the zinc binding domain of EcoRNAP β′ subunit simultaneously. Results from fluorescent polarization assays indicate the specific interactions between SspA and σ70 region 4 confer its σ selectivity, thereby avoiding its interactions with σs or other alternative σ factors. In addition, results from in vitro transcription assays verify that SspA inhibits transcription probably through suppressing promoter escape. Together, the results here provide a foundation for understanding the unique physiological function of SspA in transcription regulation in bacteria.

In vitro transcription of ribosomal RNA on phage λrifd 18 DNA

1978 ◽  
Vol 518 (2) ◽  
pp. 257-266 ◽  
Author(s):  
Ibolya Kiss ◽  
Krystina Slaska ◽  
János Sümegi ◽  
Andor Udvardy ◽  
Pál Venetianer
Keyword(s):  
Ribosomal Rna ◽  
In Vitro Transcription

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.

scholarly journals Substitutions in Bacteriophage T4 AsiA andEscherichia coli ς70 That Suppress T4motA Activation Mutations

2001 ◽  
Vol 183 (7) ◽  
pp. 2289-2297 ◽  
Author(s):  
Marco P. Cicero ◽  
Meghan M. Sharp ◽  
Carol A. Gross ◽  
Kenneth N. Kreuzer
Keyword(s):  
Rna Polymerase ◽  
Burst Size ◽  
Bacteriophage T4 ◽  
In Vitro Transcription ◽  
Positive Control ◽  
Plaque Morphology ◽  
Mutant Proteins ◽  
E Coli

ABSTRACT Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the ς70 subunit. AmotA positive control (pc) mutant, motA-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five ς70 mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione S-transferase fusions of the five mutant ς70 proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The ς70 substitutions affected the 4.2 region, which binds the −35 sequence of E. coli promoters. In the presence of E. coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E. colipromoters. This defect could not be explained solely by a disruption in −35 recognition since similar results were obtained with extended −10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support E. coli growth.

scholarly journals Construction of a synthetic messenger RNA encoding a membrane protein.

1983 ◽  
Vol 96 (5) ◽  
pp. 1464-1469 ◽  
Author(s):  
J L Rubenstein ◽  
T G Chappell
Keyword(s):  
G Protein ◽  
Messenger Rna ◽  
In Vitro Transcription ◽  
Membrane Insertion ◽  
Wheat Germ Extract ◽  
E Coli ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Synthetic Mrna

We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV-infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes.

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 Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

2021 ◽  
Author(s):  
Michela Notarangelo ◽  
Alessandro Quattrone ◽  
Massimo Pizzato ◽  
Sheref S. Mansy ◽  
O. Duhan Toparlak
Keyword(s):  
Room Temperature ◽  
Colorimetric Detection ◽  
In Vitro Transcription ◽  
Viral Rna ◽  
Rna Sequences ◽  
Rna Detection ◽  
E Coli ◽  
Free Gene ◽  
Cell Free Protein Synthesis

We report colorimetric detection of SARS-CoV-2 viral RNA by an in vitro transcription/translation assay with crude E. coli extracts at room temperature, with the aid of body heat. Clinically-relevant concentrations of viral RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved by toehold-switch-mediated riboregulatory elements that are specific to viral RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZ-ω) with cell-free expressed LacZ-α, using an X-gal analogue as a substrate. The estimated cost of single reaction is less than 1 euro/test, which may facilitate diagnostic kit accessibility in developing countries.

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