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Controlling the motor activity of a transcription-repair coupling factor: autoinhibition and the role of RNA polymerase.

ABSTRACT: Motor proteins that couple ATP hydrolysis to movement along nucleic acids play a variety of essential roles in DNA metabolism. Often these enzymes function as components of macromolecular complexes, and DNA translocation by the motor protein drives movement of other components of the complex. In order to understand how the activity of motor proteins is regulated within multi-protein complexes we have studied the bacterial transcription-repair coupling factor, Mfd, which is a helicase superfamily 2 member that binds to RNA polymerase (RNAP) and removes stalled transcription complexes from DNA. Using an oligonucleotide displacement assay that monitors protein movement on double-stranded DNA we show that Mfd has little motor activity in isolation, but exhibits efficient oligonucleotide displacement activity when bound to a stalled transcription complex. Deletion of the C-terminal domain of Mfd increases the ATPase activity of the protein and allows efficient oligo-displacement in the absence of RNAP. Our results suggest that an autoinhibitory domain ensures the motor activity of Mfd is only functional within the correct macromolecular context: recruitment of Mfd to a stalled transcription complex relieves the autoinhibition and unmasks the motor activity.

SUBMITTER: Smith AJ

PROVIDER: S-EPMC1874598 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Controlling the motor activity of a transcription-repair coupling factor: autoinhibition and the role of RNA polymerase.

Smith Abigail J AJ Szczelkun Mark D MD Savery Nigel J NJ

Nucleic acids research 20070228 6

Motor proteins that couple ATP hydrolysis to movement along nucleic acids play a variety of essential roles in DNA metabolism. Often these enzymes function as components of macromolecular complexes, and DNA translocation by the motor protein drives movement of other components of the complex. In order to understand how the activity of motor proteins is regulated within multi-protein complexes we have studied the bacterial transcription-repair coupling factor, Mfd, which is a helicase superfamily ...[more]

PMID: 17329375

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Project description:In growing bacterial cells, the global reorganization of transcription is associated with alterations of RNA polymerase composition and the superhelical density of the DNA. However, the existence of any regulatory device coordinating these changes remains elusive. Here we show that in an exponentially growing Escherichia coli rpoZ mutant lacking the polymerase ? subunit, the impact of the E?(38) holoenzyme on transcription is enhanced in parallel with overall DNA relaxation. Conversely, overproduction of ?(70) in an rpoZ mutant increases both overall DNA supercoiling and the transcription of genes utilizing high negative superhelicity. We further show that transcription driven by the E?(38) and E?(70) holoenzymes from cognate promoters induces distinct superhelical densities of plasmid DNA in vivo. We thus demonstrate a tight coupling between polymerase holoenzyme composition and the supercoiling regimen of genomic transcription. Accordingly, we identify functional clusters of genes with distinct ? factor and supercoiling preferences arranging alternative transcription programs sustaining bacterial exponential growth. We propose that structural coupling between DNA topology and holoenzyme composition provides a basic regulatory device for coordinating genome-wide transcription during bacterial growth and adaptation. IMPORTANCE Understanding the mechanisms of coordinated gene expression is pivotal for developing knowledge-based approaches to manipulating bacterial physiology, which is a problem of central importance for applications of biotechnology and medicine. This study explores the relationships between variations in the composition of the transcription machinery and chromosomal DNA topology and suggests a tight interdependence of these two variables as the major coordinating principle of gene regulation. The proposed structural coupling between the transcription machinery and DNA topology has evolutionary implications and suggests a new methodology for studying concerted alterations of gene expression during normal and pathogenic growth both in bacteria and in higher organisms.

Dataset Information

Controlling the motor activity of a transcription-repair coupling factor: autoinhibition and the role of RNA polymerase.

Publications

Controlling the motor activity of a transcription-repair coupling factor: autoinhibition and the role of RNA polymerase.

Similar Datasets

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