scholarly journals Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs

2020 ◽  
Author(s):  
Anthony L. Shiver ◽  
Hendrik Osadnik ◽  
Jason M. Peters ◽  
Rachel A. Mooney ◽  
Peter I. Wu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Structure Function ◽  
Protein Complexes ◽  
Chemical Genetic ◽  
Trade Offs ◽  
Bacterial Rna ◽  
Genes Encoding ◽  
Mutant Phenotypes ◽  
Polymerase Mutants

AbstractThe multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated the inner workings of RNAP, and mutations in genes encoding RNAP drive adaptation of Escherichia coli to many health- and industry-relevant environments, yet a paucity of systematic analyses has hampered our understanding of the fitness benefits and trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and illuminate a connection between transcription and cell division. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships in vivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.

scholarly journals Chemical-genetic interrogation of RNA polymerase mutants reveals structure-function relationships and physiological tradeoffs

2021 ◽  
Vol 81 (10) ◽  
pp. 2201-2215.e9
Author(s):  
Anthony L. Shiver ◽  
Hendrik Osadnik ◽  
Jason M. Peters ◽  
Rachel A. Mooney ◽  
Peter I. Wu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Structure Function ◽  
Chemical Genetic ◽  
Polymerase Mutants

scholarly journals Substitution of a Highly Conserved Histidine in the Escherichia coli Heat Shock Transcription Factor, σ32, Affects Promoter Utilization In Vitro and Leads to Overexpression of the Biofilm-Associated Flu Protein In Vivo

2007 ◽  
Vol 189 (23) ◽  
pp. 8430-8436 ◽  
Author(s):  
Olga V. Kourennaia ◽  
Pieter L. deHaseth
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Stable Complex ◽  
Tryptophan Residue ◽  
Specific Sequence ◽  
Bacterial Rna

ABSTRACT The heat shock sigma factor (σ32 in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of σ32 is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, σ70. This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing σ32, implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in σ32 impedes recovery from heat shock (exposure to 42°C), and it also leads to overexpression at lower temperatures (30°C) of the Flu protein, which is associated with biofilm formation.

scholarly journals Genetic interaction mapping informs integrative structure determination of protein complexes

Science ◽  
2020 ◽  
Vol 370 (6522) ◽  
pp. eaaz4910 ◽  
Author(s):  
Hannes Braberg ◽  
Ignacia Echeverria ◽  
Stefan Bohn ◽  
Peter Cimermancic ◽  
Anthony Shiver ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Structure Determination ◽  
Genetic Interaction ◽  
Protein Complexes ◽  
Point Mutations ◽  
Genetic Interactions ◽  
Cellular Functions ◽  
Cross Links

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.

scholarly journals Modeling neurodegeneration in Caenorhabditiselegans

2020 ◽  
Vol 13 (10) ◽  
pp. dmm046110
Author(s):  
Kim A. Caldwell ◽  
Corey W. Willicott ◽  
Guy A. Caldwell
Keyword(s):  
Neurodegenerative Diseases ◽  
Drug Targets ◽  
Fluorescent Proteins ◽  
Critical Threshold ◽  
C Elegans ◽  
Chemical Genetic ◽  
Modifying Factors ◽  
Genes Encoding ◽  
The Impact

ABSTRACTThe global burden of neurodegenerative diseases underscores the urgent need for innovative strategies to define new drug targets and disease-modifying factors. The nematode Caenorhabditis elegans has served as the experimental subject for multiple transformative discoveries that have redefined our understanding of biology for ∼60 years. More recently, the considerable attributes of C. elegans have been applied to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease and Huntington's disease. Transgenic nematodes with genes encoding normal and disease variants of proteins at the single- or multi-copy level under neuronal-specific promoters limits expression to select neuronal subtypes. The anatomical transparency of C. elegans affords the use of co-expressed fluorescent proteins to follow the progression of neurodegeneration as the animals age. Significantly, a completely defined connectome facilitates detailed understanding of the impact of neurodegeneration on organismal health and offers a unique capacity to accurately link cell death with behavioral dysfunction or phenotypic variation in vivo. Moreover, chemical treatments, as well as forward and reverse genetic screening, hasten the identification of modifiers that alter neurodegeneration. When combined, these chemical-genetic analyses establish critical threshold states to enhance or reduce cellular stress for dissecting associated pathways. Furthermore, C. elegans can rapidly reveal whether lifespan or healthspan factor into neurodegenerative processes. Here, we outline the methodologies employed to investigate neurodegeneration in C. elegans and highlight numerous studies that exemplify its utility as a pre-clinical intermediary to expedite and inform mammalian translational research.

scholarly journals Structural and functional features of Crl proteins and identification of conserved surface residues required for interaction with the RpoS/σS subunit of RNA polymerase

2014 ◽  
Vol 463 (2) ◽  
pp. 215-224 ◽  
Author(s):  
Paola Cavaliere ◽  
Fabienne Levi-Acobas ◽  
Claudine Mayer ◽  
Frederick A. Saul ◽  
Patrick England ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
In Vitro Studies ◽  
Conserved Residues ◽  
Bacterial Rna ◽  
Functional Features ◽  
Flexible Arms ◽  
Binding Determinants

The Crl protein binds and activates the σS subunit of bacterial RNA polymerase. In vivo and in vitro studies revealed that the σS-binding determinants of Crl lie in conserved residues located in a cavity enclosed by flexible arms.

scholarly journals No correlation between binding of glucocorticosteroids to specific cytoplasmic proteins in vivo and enzyme induction in the rat liver

1983 ◽  
Vol 212 (2) ◽  
pp. 305-312 ◽  
Author(s):  
H Grote ◽  
J Voigt ◽  
C E Sekeris
Keyword(s):  
Rna Polymerase ◽  
Rat Liver ◽  
Enzyme Induction ◽  
Protein Complexes ◽  
Dose Dependence ◽  
Tyrosine Aminotransferase ◽  
Biologically Active ◽  
Cytoplasmic Proteins ◽  
Stimulation Of

Time- and dose-dependence of the formation of the different cytoplasmic hormone-protein complexes were studied in the rat liver after administration in vivo of [3H]cortisol or [3H]dexamethasone and compared with the stimulation of RNA polymerase B and induction of tyrosine aminotransferase and tryptophan oxygenase. No correlation could be found between formation in vivo of any of the five cytoplasmic hormone-protein complexes found and stimulation of RNA polymerase B activity or enzyme induction. After administration of [3H]cortisol, different metabolites of cortisol could be demonstrated in the isolated hormone-protein complexes. No time- or dose-dependence of the metabolite patterns could be observed after application of hormone doses that were in the range of the biologically active doses. After administration of [3H]dexamethasone, the same hormone-protein complexes were observed, which contained, however, the injected steroid instead of metabolites. These results seem to indicate that the cytoplasmic binding components present in the rat liver are enzymes involved in the metabolism of the glucocorticosteroids and that dexamethasone binds to these enzymes as a substrate analogue.

New Antimicrobials Targeting Bacterial RNA Polymerase Holoenzyme Assembly Identified with an in Vivo BRET-Based Discovery Platform

2019 ◽  
Vol 14 (8) ◽  
pp. 1727-1736 ◽  
Author(s):  
Sara Sartini ◽  
Elisabetta Levati ◽  
Martina Maccesi ◽  
Matteo Guerra ◽  
Gilberto Spadoni ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Bacterial Rna ◽  
Rna Polymerase Holoenzyme

scholarly journals N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases

2002 ◽  
Vol 184 (18) ◽  
pp. 4952-4961 ◽  
Author(s):  
S. H. Willis ◽  
K. M. Kazmierczak ◽  
R. H. Carter ◽  
L. B. Rothman-Denes
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Sequence Similarity ◽  
Rna Polymerases ◽  
Transcriptional Analysis ◽  
Amino Acid Residues ◽  
Genes Encoding ◽  
Single Subunit

ABSTRACT Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII). Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3. These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases. The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed. Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro.

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