Translocation of DNA-Dependent E. coli RNA Polymerase During RNA Synthesis

1997 ◽  
pp. 151-177 ◽  
Author(s):  
H. Heumann ◽  
E. Zaychikov ◽  
L. Denissova ◽  
T. Hermann
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
E Coli

scholarly journals Effect of DNA-interacting drugs on phage T7 RNA polymerase.

1998 ◽  
Vol 45 (1) ◽  
pp. 127-132 ◽  
Author(s):  
M Piestrzeniewicz ◽  
K Studzian ◽  
D Wilmańska ◽  
G Płucienniczak ◽  
M Gniazdowski
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
T7 Rna Polymerase ◽  
Distamycin A ◽  
E Coli ◽  
Phage T7 ◽  
Drug Dependent ◽  
Dna Complex ◽  
Kinetics Of

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

scholarly journals Transcription of bacteriophage T4 deoxyribonucleic acid in vitro

1969 ◽  
Vol 115 (3) ◽  
pp. 353-361 ◽  
Author(s):  
John O. Bishop ◽  
Forbes W. Robertson
Keyword(s):  
Rna Polymerase ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Bacteriophage T4 ◽  
Rna Sequences ◽  
Experimental Conditions ◽  
E Coli ◽  
New Methods

1. RNA was synthesized in vitro from a template of bacteriophage T4 DNA, in the presence of Mn2+. A comparison was made of the RNA synthesized by purified RNA polymerase from two sources, Micrococcus lysodeikticus and Escherichia coli; these are referred to as Micrococcus cRNA and E. coli cRNA respectively (where cRNA indicates RNA synthesized in vitro by using purified RNA polymerase and a DNA primer). 2. Both types of RNA were self-complementary as judged by resistance to digestion with ribonuclease after self-annealing, Micrococcus cRNA being more self-complementary (40%) than was E. coli cRNA (30%). The cRNA was found to be much less self-complementary if Mg2+ was present during RNA synthesis instead of Mn2+. 3. Micrococcus cRNA hybridized with a larger part of bacteriophage T4 DNA than did E. coli cRNA. The E. coli cRNA competed with only part (70%) of the Micrococcus cRNA in hybridization-competition experiments. It is concluded that more sequences of bacteriophage T4 DNA are transcribed by Micrococcus polymerase than by E. coli polymerase. 4. The RNA sequences synthesized by Micrococcus RNA polymerase but not by E. coli RNA polymerase are shown by hybridization competition to compete with specifically late bacteriophage T4 messenger RNA sequences. The relevance of this finding to the control of transcription is discussed. 5. In an Appendix, new methods are described for the analysis of hybridization-saturation and -competition experiments. Particular attention is paid to the effects produced if different RNA sequences are present at different relative concentrations. 6. By using cRNA isolated from an enzymically synthesized DNA–RNA hybrid, it is estimated that, of the DNA that is complementary to cRNA, only about half can become hybridized with cRNA under the experimental conditions used.

scholarly journals Effect of multiple copies ofrpoBCon the rate of RNA synthesis inEscherichia coli

1986 ◽  
Vol 48 (2) ◽  
pp. 61-64 ◽  
Author(s):  
Elena C. Guzman ◽  
Alfonso Jimenez-Sanchez
Keyword(s):  
Dna Replication ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Synthetic Activity ◽  
Gene Products ◽  
Replication Forks ◽  
E Coli ◽  
Multiple Copies ◽  
Autogenous Regulation ◽  
Rate Of Movement

SummaryThe cloning of therpoBandrpoCgenes in a high copy number vector inE. coliincreased the amount of the encoded gene products, the β and β′ subunits of RNA polymerase. However, this unexpectedly caused a 30–50% decrease in RNA synthetic activity which alternatively induced a reduction of growth rate and enlargement of cell size, and decreased the DNA replication time. The results can be explained by autogenous regulation of the RNA polymerase genes by the ββ′ subunits. A relation between the decrease in number of transcription units and the observed higher rate of movement of DNA replication forks is discussed.

scholarly journals Antimicrobial Properties and Mode of Action of the Pyrrothine Holomycin

2001 ◽  
Vol 45 (2) ◽  
pp. 532-539 ◽  
Author(s):  
Brunello Oliva ◽  
Alexander O'Neill ◽  
Jenny M. Wilson ◽  
Peter J. O'Hanlon ◽  
Ian Chopra
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Antimicrobial Properties ◽  
Stringent Response ◽  
Active Species ◽  
Chain Elongation ◽  
Whole Cells ◽  
E Coli ◽  
Eukaryotic Microorganisms ◽  
Kinetics Of

ABSTRACT Holomycin, a member of the pyrrothine class of antibiotics, displayed broad-spectrum antibacterial activity, inhibiting a variety of gram-positive and gram-negative bacteria, with the exception ofEnterobacter cloacae, Morganella morganii, andPseudomonas aeruginosa. The antibiotic lacked activity against the eukaryotic microorganisms Saccharomyces cerevisiae and Candida kefyr. Holomycin exhibited a bacteriostatic response against Escherichia coli that was associated with rapid inhibition of RNA synthesis in whole cells. Inhibition of RNA synthesis could have been a secondary consequence of inhibiting tRNA aminoacylation, thereby inducing the stringent response. However, the levels of inhibition of RNA synthesis by holomycin were similar in a stringent and relaxed pair of E. coli strains that were isogenic except for the deletion of therelA gene. This suggests that inhibition of RNA synthesis by holomycin could reflect direct inhibition of DNA-dependent RNA polymerase. Examination of the effects of holomycin on the kinetics of the appearance of β-galactosidase in induced E. colicells was also consistent with inhibition of RNA polymerase at the level of RNA chain elongation. However, holomycin only weakly inhibitedE. coli RNA polymerase in assays using synthetic poly(dA-dT) and plasmid templates. Furthermore, inhibition of RNA polymerase was observed only at holomycin concentrations in excess of those required to inhibit the growth of E. coli. It is possible that holomycin is a prodrug, requiring conversion in the cell to an active species that inhibits RNA polymerase.

scholarly journals NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Matti Turtola ◽  
Georgiy A Belogurov
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Transcription Elongation ◽  
Single Nucleotide ◽  
E Coli ◽  
Transcription Bubble ◽  
Nucleotide Addition ◽  
Nascent Rna ◽  
Comprehensive Mapping

Universally conserved factors from NusG family bind at the upstream fork junction of transcription elongation complexes and modulate RNA synthesis in response to translation, processing, and folding of the nascent RNA. Escherichia coli NusG enhances transcription elongation in vitro by a poorly understood mechanism. Here we report that E. coli NusG slows Gre factor-stimulated cleavage of the nascent RNA, but does not measurably change the rates of single nucleotide addition and translocation by a non-paused RNA polymerase. We demonstrate that NusG slows RNA cleavage by inhibiting backtracking. This activity is abolished by mismatches in the upstream DNA and is independent of the gate and rudder loops, but is partially dependent on the lid loop. Our comprehensive mapping of the upstream fork junction by base analogue fluorescence and nucleic acids crosslinking suggests that NusG inhibits backtracking by stabilizing the minimal transcription bubble.

scholarly journals Asymmetric RNA synthesis in vitro: heterologous DNA-enzyme systems; E. coli RNA polymerase.

1965 ◽  
Vol 53 (5) ◽  
pp. 1140-1147 ◽  
Author(s):  
A. J. Colvill ◽  
L. C. Kanner ◽  
G. P. Tocchini-Valentini ◽  
M. T. Sarnat ◽  
E. P. Geiduschek
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
E Coli ◽  
Enzyme Systems

scholarly journals Action of intact AP (apurinic/apyrimidinic) sites and AP sites associated with breaks on the transcription of T7 coliphage DNA by Escherichia coli RNA polymerase

1985 ◽  
Vol 229 (1) ◽  
pp. 173-181 ◽  
Author(s):  
P A Flamée ◽  
W G Verly
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Alkylating Agents ◽  
Experimental Conditions ◽  
E Coli ◽  
Template Strand ◽  
Ap Sites ◽  
Ap Site

The effect of apurinic/apyrimidinic (AP) sites in DNA on RNA and protein synthesis was studied in vitro using T7 coliphage DNA. Initiation of RNA synthesis by Escherichia coli RNA polymerase was synchronized and heparin was used to prevent reinitiation. When the T7 DNA contained AP sites, the rate of RNA synthesis was decreased but it remained higher than the values calculated on the assumption that an AP site in the transcribed strand is a complete block to the enzyme progression. Moreover, after the time taken by an unimpeded enzyme to go from promoter to terminator, the rate of RNA synthesis remained elevated and the number of complete RNA molecules (7000 nucleotides) continued to increase for some time. These results suggest that, if the E. coli RNA polymerase is stopped by an AP site, most often, after a pause, the enzyme resumes elongation of the RNA chain which is continuous over the AP site. Sometimes however, RNA synthesis is definitively interrupted during the pause; the probability of interruption has been estimated to be 0.3 in our experimental conditions. When a nick is placed 5′ to the AP site by an AP endonuclease, the results are similar: most often, the RNA chain is synthesized without interruption past the nick in the template strand. The pause of the E. coli RNA polymerase at this combined lesion appears to be shorter than when the AP site is intact. To investigate whether a nucleotide is placed in the RNA chain in front of the AP site in the template strand by E. coli RNA polymerase, RNA synthesis was taken to completion before using this RNA for protein synthesis and measuring the activity of gene-1 product, T7 RNA polymerase. The result suggests that, after pausing, the E. coli RNA polymerase places a nucleotide in the RNA chain when passing over an AP site. The mechanism of the delayed lethality of T7 coliphages treated with monofunctional alkylating agents, which is due to the appearance of AP sites, is discussed.

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