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

ABSTRACT: The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness 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 demonstrate that some RNA polymerase mutants both decrease average cell length and prevent killing by cell-wall targeting antibiotics. 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.

SUBMITTER: Shiver AL

PROVIDER: S-EPMC8484514 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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

Shiver Anthony L AL Osadnik Hendrik H Peters Jason M JM Mooney Rachel A RA Wu Peter I PI Henry Kemardo K KK Braberg Hannes H Krogan Nevan J NJ Hu James C JC Landick Robert R Huang Kerwyn Casey KC Gross Carol A CA

Molecular cell 20210501 10

The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We dis ...[more]

PMID: 34019789

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Project description:Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-? resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 ? from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-? interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A.The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses.

Dataset Information

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

Publications

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

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