Identification of the amatoxin-binding subunit of RNA polymerase B by affinity labeling experiments. Subunit B 3 - the true amatoxin receptor protein of multiple RNA polymerase B

Biochemistry ◽  
1976 ◽  
Vol 15 (16) ◽  
pp. 3480-3484 ◽  
Author(s):  
Ortfried G. Brodner ◽  
Theodor Wieland
Keyword(s):  
Rna Polymerase ◽  
Receptor Protein ◽  
Affinity Labeling ◽  
Subunit B ◽  
Binding Subunit

scholarly journals Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the α subunit of RNA polymerase at Class I promoters

1999 ◽  
Vol 337 (3) ◽  
pp. 415-423 ◽  
Author(s):  
Emma C. LAW ◽  
Nigel J. SAVERY ◽  
Stephen J. W. BUSBY
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Class I ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Α Subunit ◽  
Terminal Domain ◽  
Up Elements ◽  
Camp Receptor

The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters. At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase α subunit (αCTD). Since αCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions. We previously showed that, at these promoters, the CRP–αCTD interaction and the CRP–UP element interaction contribute independently and additively to transcription initiation. In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5. This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two αCTDs of RNA polymerase are not optimally positioned. Two experiments to test this hypothesis are presented.

scholarly journals Interdependence of Activation at rhaSR by Cyclic AMP Receptor Protein, the RNA Polymerase Alpha Subunit C-Terminal Domain, and RhaR

2000 ◽  
Vol 182 (23) ◽  
pp. 6774-6782 ◽  
Author(s):  
Carolyn C. Holcroft ◽  
Susan M. Egan
Keyword(s):  
Cyclic Amp ◽  
Rna Polymerase ◽  
Synergistic Effects ◽  
Receptor Protein ◽  
Mobility Shift ◽  
Α Subunit ◽  
Subunit C ◽  
Cyclic Amp Receptor Protein ◽  
Terminal Domain ◽  
Full Activation

ABSTRACT The Escherichia coli rhaSR operon encodes two AraC family transcription activators, RhaS and RhaR, and is activated by RhaR in the presence of l-rhamnose. β-Galactosidase assays of various rhaS-lacZ promoter fusions combined with mobility shift assays indicated that a cyclic AMP receptor protein (CRP) site located at −111.5 is also required for full activation of rhaSR expression. To address the mechanisms of activation by CRP and the RNA polymerase α-subunit C-terminal domain (α-CTD) at rhaSR, we tested the effects of alanine substitutions in CRP activating regions 1 and 2, overexpression of a truncated version of α (α-Δ235), and alanine substitutions throughout α-CTD. We found that DNA-contacting residues in α-CTD are required for full activation, and for simplicity, we discuss α-CTD as a third activator of rhaSR. CRP and RhaR could each partially activate transcription in the absence of the other two activators, and α-CTD was not capable of activation alone. In the case of CRP, this suggests that this activation involves neither an α-CTD interaction nor cooperative binding with RhaR, while in the case of RhaR, this suggests the likelihood of direct interactions with core RNA polymerase. We also found that CRP, RhaR, and α-CTD each have synergistic effects on activation by the others, suggesting direct or indirect interactions among all three. We have some evidence that the α-CTD–CRP and α-CTD–RhaR interactions might be direct. The magnitude of the synergistic effects was usually greater with just two activators than with all three, suggesting possible redundancies in the mechanisms of activation by CRP, α-CTD, and RhaR.

scholarly journals Molecular Docking Analysis of the T450A Mutation of the Gene rpoB Mycobacterium leprae from Leprosy Patients in Papua, West Papua and North Maluku, Indonesia

2021 ◽  
pp. 3578-3584
Author(s):  
Yustinus Maladan ◽  
Hana Krismawati ◽  
Tri Wahyuni ◽  
Hotma Martogi Lorensi Hutapea ◽  
Muhammad Fajri Rokhmad ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Rna Polymerase ◽  
Binding Affinity ◽  
Rpob Gene ◽  
Ramachandran Plot ◽  
Docking Analysis ◽  
West Papua ◽  
Leprosy Patients ◽  
Subunit B ◽  
Molecular Docking Analysis

Leprosy persists to be a health problem in Indonesia, especially in the provinces of North Maluku, West Papua and Papua. Early diagnosis and complete treatment with multidrug therapy (MDT) remain the key strategy for reducing the disease burden. One of the major components of MDT is rifampicin which in certain cases in several countries, M. leprae resistance to this drug issue has been reported albeit only a few. This research aimed to detect and analyze polymorphism in M. leprae rpoB gene that was isolated from leprosy patients in three provinces: North Maluku Province, West Papua Province and Papua Province, Indonesia. The identification of mutations in the M. leprae rpoB gene was carried out by aligning the results of DNA sequencing with the reference strain. The 3D structure of rpoB was derived using the Swiss Model. The T450A, S456L, and H451Y variants of RNA Polymerase B subunits were constructed using FoldX based on the wild-type structure. The structures were repaired, and protein stability was evaluated using foldX under the Yasara viewer. The QC of the rpoB M. leprae homology models was conducted with Ramachandran Plot modeling using PROCHECK. The difference in binding affinity between native protein and T450A, S456L, and H45I variants were analyzed using molecular docking. rpoB gene of M. leprae contains a mutation found in nucleotide of 1348 bp. The mutation triggered the conversion of the amino acid Threonine to Alanine in the amino acid to 450 rpoB subunit B. The structure of 3D RNA Polymerase Subunit B was constructed using rpoB Mycobacterium tuberculosis with PDB code 5UH5 as template. According to Ramachandran Plot, the percentage of residues in the most favored regions are 91.9%, and there was no significant number of residues in the disallowed regions. The results of molecular docking showed that the T450A variant had the same binding affinity with the native protein which was -8.9 kcal. Binding affinity on the S456L and H451Y variants increased by -7.3 kcal and -8.2 kcal, respectively. According to Molecular Docking analysis, T450A variant did not affect the energy binding between RNA polymerase and rifampicin.

scholarly journals Fine-structure genetics of ama-1, an essential gene encoding the amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans.

Genetics ◽  
1988 ◽  
Vol 120 (2) ◽  
pp. 423-434
Author(s):  
A M Bullerjahn ◽  
D L Riddle
Keyword(s):  
Fine Structure ◽  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Essential Gene ◽  
Resistance Mutation ◽  
Lethal Mutation ◽  
Intragenic Recombination ◽  
Lethal Mutations ◽  
Binding Subunit

Abstract A fine-structure genetic map has been constructed for ama-1 IV, an essential gene in Caenorhabditis elegans encoding the amanitin-binding subunit of RNA polymerase II. Sixteen EMS-induced recessive-lethal mutations have been positioned in the gene by determining their intragenic recombination frequencies with m118, a mutation that confers dominant resistance to alpha-amanitin. The 16 mutants, all isolated in the ama-1(m118) background, include 13 that are early larval lethals, and three that are mid-larval lethals, at 25 degrees. Six of the mutants exhibit temperature-dependence in the severity of their phenotype. Intragenic recombination between the lethal site and the parental resistance mutation was detected by means of resistance to amanitin. Recombinants were detected at frequencies as low as 2 X 10(-6). The segregation of the closely linked flanking markers, unc-17 and unc-5, revealed whether the lethal mutation was to the left or the right of m118. By adding the distances between the extreme left and right mutations, the ama-1 gene is estimated to be 0.011 map unit long, with m118 positioned 0.004 map unit from the left-most lethal mutation. To order the lethal mutations with respect to each other, viable heteroallelic strains were constructed using the free duplication, mDp1[unc-17(e113) dpy-13(+) ama-1(+)]. The heteroallelic strains were sensitive to amanitin, and recombination events between the lethal mutations were specifically selected by means of the dominant amanitin resistance encoded on the recombinant chromosome. The segregation of outside markers revealed the left-right order of the lethal mutations. The position of mutations within the gene is nonrandom. Functional domains of the ama-1 gene indicated by the various lethal phenotypes are discussed.

Affinity labeling the bovine gallbladder cholecystokinin receptor using a battery of probes

1988 ◽  
Vol 255 (5) ◽  
pp. G579-G586
Author(s):  
B. Schjoldager ◽  
S. P. Powers ◽  
L. J. Miller
Keyword(s):  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Peptide Hormone ◽  
Molecular Probes ◽  
Affinity Labeling ◽  
Molecular Heterogeneity ◽  
Cck Receptors ◽  
Amino Terminal ◽  
Binding Subunit

Although the gallbladder was the first recognized target of the peptide hormone cholecystokinin (CCK) and is a physiologically important target, only one preliminary report of the biochemical characterization of this receptor exists. Recently, a series of molecular probes for the affinity labeling of different domains of the pancreatic CCK receptor have been developed. In this work we report the application of several of those probes toward the biochemical characterization of the bovine gallbladder muscularis receptor. These include "long" (125I-Bolton-Hunter-CCK-33) and "short" (125I-D-Tyr-Gly-[Nle28,31)CCK-(26-33)]) probes chemically cross-linkable through their amino-terminal amino groups and monofunctional probes with their photolabile moieties at their amino terminus (2-diazo-3,3,3-trifluoropropionyl-125I-D-Tyr-Gly-[(Nle28,31) CCK-(26-33)]) and carboxyl terminus (125I-D-Tyr-Gly-[(Nle28,31,pNO2-Phe33)CCK-(26-33)]), that span the receptor-binding region. Each of these bound specifically and saturably to a preparation enriched in plasma membranes from bovine gallbladder muscularis (mean inhibitor constants: 5.2, 1.1, 0.8, and 1.8 nM, respectively). A major relative molecular weight (Mr) 70,000-85,000 band was specifically and reproducibly labeled with the appropriate apparent affinity by each of the probes, whereas labeling of minor bands of Mr 40,000-50,000, Mr 92,000, Mr 120,000, and Mr 200,000 was dependent on cross-linker type or concentration. These observations support the identification of the Mr 70,000-85,000 protein as the bovine gallbladder CCK-binding subunit and, since this is a different size from the pancreatic CCK-binding subunit, provide biochemical evidence for molecular heterogeneity of peripheral CCK receptors.

scholarly journals Cyclic AMP Receptor Protein-Dependent Activation of the Escherichia coli acsP2 Promoter by a Synergistic Class III Mechanism

2003 ◽  
Vol 185 (17) ◽  
pp. 5148-5157 ◽  
Author(s):  
Christine M. Beatty ◽  
Douglas F. Browning ◽  
Stephen J. W. Busby ◽  
Alan J. Wolfe
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class I ◽  
Receptor Protein ◽  
Class Iii ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Rna Polymerase Holoenzyme

ABSTRACT The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by “focusing” RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions −69.5 and −122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the α subunit of RNA polymerase holoenzyme (α-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the α-CTD. Other surface-exposed residues in the α-CTD contributed to acs transcription, suggesting that the α-CTD may interact with at least one protein other than CRP.

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