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Title: RNA polymerase motions during promoter melting

Authors:
ORCiD logo; ORCiD logo; ORCiD logo; ORCiD logo; ORCiD logo; ; ORCiD logo; ; ORCiD logo; ORCiD logo
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
NIGMS
OSTI Identifier:
1368297
Resource Type:
Journal Article
Resource Relation:
Journal Name: Science; Journal Volume: 356; Journal Issue: 6340
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Feklistov, Andrey, Bae, Brian, Hauver, Jesse, Lass-Napiorkowska, Agnieszka, Kalesse, Markus, Glaus, Florian, Altmann, Karl-Heinz, Heyduk, Tomasz, Landick, Robert, and Darst, Seth A. RNA polymerase motions during promoter melting. United States: N. p., 2017. Web. doi:10.1126/science.aam7858.
Feklistov, Andrey, Bae, Brian, Hauver, Jesse, Lass-Napiorkowska, Agnieszka, Kalesse, Markus, Glaus, Florian, Altmann, Karl-Heinz, Heyduk, Tomasz, Landick, Robert, & Darst, Seth A. RNA polymerase motions during promoter melting. United States. doi:10.1126/science.aam7858.
Feklistov, Andrey, Bae, Brian, Hauver, Jesse, Lass-Napiorkowska, Agnieszka, Kalesse, Markus, Glaus, Florian, Altmann, Karl-Heinz, Heyduk, Tomasz, Landick, Robert, and Darst, Seth A. Thu . "RNA polymerase motions during promoter melting". United States. doi:10.1126/science.aam7858.
@article{osti_1368297,
title = {RNA polymerase motions during promoter melting},
author = {Feklistov, Andrey and Bae, Brian and Hauver, Jesse and Lass-Napiorkowska, Agnieszka and Kalesse, Markus and Glaus, Florian and Altmann, Karl-Heinz and Heyduk, Tomasz and Landick, Robert and Darst, Seth A.},
abstractNote = {},
doi = {10.1126/science.aam7858},
journal = {Science},
number = 6340,
volume = 356,
place = {United States},
year = {Thu May 25 00:00:00 EDT 2017},
month = {Thu May 25 00:00:00 EDT 2017}
}
  • Complexes between Bacillus subtilus RNA polymerase and /sup 32/P-labeled DNA were irradiated with UV light and digested with nuclease; electrophoresis and autoradiography were used to identify the polymerase subunits cross-linked to DNA. These experiments showed: 1) that cross-linkage of promoter complexes yielded predominantly the beta and sigma subunits; 2) that beta, beta', and sigma were detected in non-promoter complexes; 3) that addition of the delta subunit or high concentrations of NaCl decreased cross-linkage of all subunits, especially the cross-linkage of the sigma subunit in non-promoter complexes and the binding of polymerase at DNA ends; 4) that different patterns of cross-linkagemore » were obtained at 0 degrees C (conditions favoring the formation of closed complexes) and 37 degrees C (conditions favoring the formation of open complexes); and 5) predominantly beta and possibly alpha were cross-linked by irradiation of core-DNA complexes whereas similar experiments with core-delta complexed to DNA showed the efficient cross-linkage of beta' and beta.« less
  • Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiationmore » codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested.« less
  • By using a protein-DNA cross-linking method, 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 vapormore » 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.« less
  • Three promoters direct transcription of the sigA (rpoD) operon in Bacillus subtilis. Promoters P1 and P2 are used during the exponential growth phase, whereas P3 is used only during the stationary phase. We examined the use of these promoters in promoter-probe plasmids and found that expression from P3 was prevented by a mutation in spoOH, which encodes the secondary RNA polymerase sigma factor sigma/sup H/. Moreover, we found that sigma/sup H/-containing RNA polymerase efficiently and accurately used the P3 promoter in vitro. Evidently, this operon, which is essential for exponential growth, is transcribed during the early phase of sporulation bymore » this secondary form of RNA polymerase. Comparison of the nucleotide sequences of the P3 promoter and the spoVG promoter, which also is used by ..beta../sup H/-RNA polymerase, revealed sequences at the -10 and -35 regions of these promoters that may signal recognition of promoters by sigma/sup H/-RNA polymerase.« less
  • No abstract prepared.