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ATP-dependent nucleic acid helicases and translocases play essential roles in many aspects of DNA and RNA biology. In order to ensure that these proteins act only in specific contexts, their activity is often regulated by intramolecular contacts and interaction with partner proteins. We have studied the bacterial Mfd protein, which is an ATP-dependent DNA translocase that relocates or displaces transcription ECs in a variety of cellular contexts. When bound to RNAP, Mfd exhibits robust ATPase and DNA translocase activities, but when released from its substrate these activities are repressed by autoinhibitory interdomain contacts. In this work, we have identified an interface within the Mfd protein that is important for regulating the activity of the protein, and whose disruption permits Mfd to act indiscriminately at transcription complexes that lack the usual determinants of Mfd specificity. Our results indicate that regulation of Mfd occurs through multiple nodes, and that activation of Mfd may be a multi-stage process.

During nucleotide excision repair (NER) in bacteria the UvrC nuclease and the short oligonucleotide that contains the DNA lesion are removed from the post-incision complex by UvrD, a superfamily 1A helicase. Helicases are frequently regulated by interactions with partner proteins, and immunoprecipitation experiments have previously indicated that UvrD interacts with UvrB, a component of the post-incision complex. We examined this interaction using 2-hybrid analysis and surface plasmon resonance spectroscopy, and found that the N-terminal domain and the unstructured region at the C-terminus of UvrD interact with UvrB. We analysed the properties of a truncated UvrD protein that lacked the unstructured C-terminal region and found that it showed a diminished affinity for single-stranded DNA, but retained the ability to displace both UvrC and the lesion-containing oligonucleotide from a post-incision nucleotide excision repair complex. The interaction of the C-terminal region of UvrD with UvrB is therefore not an essential feature of the mechanism by which UvrD disassembles the post-incision complex during NER. In further experiments we showed that PcrA helicase from Bacillus stearothermophilus can also displace UvrC and the excised oligonucleotide from a post-incision NER complex, which supports the idea that PcrA performs a UvrD-like function during NER in gram-positive organisms.

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.

R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report realtime observations of cotranscriptional R-loop formation at singlemolecule resolution and propose a mechanism for their formation. We show that the bacterial Mfd protein can simultaneously interact with both elongating RNA polymerase and upstream DNA, tethering the two together and partitioning the DNA into distinct supercoiled domains. A highly negatively supercoiled domain forms in between Mfd and RNA polymerase, and compensatory positive supercoiling appears in front of the RNA polymerase and behind Mfd. The nascent RNA invades the negatively supercoiled domain and forms a stable R-loop that can drive mutagenesis. This mechanism theoretically enables any protein that simultaneously binds an actively translocating RNA polymerase and upstream DNA to stimulate R-loop formation. [ABSTRACT FROM AUTHOR]

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRPdependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase α subunit C-terminal domain (αCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase a subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within αCTD for transcription activation at a Class II CRP-dependent promoter. Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285–288 and 317. Residues 285–288 and 317 comprise a discrete 20310 Å surface on αCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between a subunits and CRP, but do not affect DNA binding by a subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in &lpha;CTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for αCTD-CRP interaction in Class I CRP-dependent transcription. [ABSTRACT FROM AUTHOR]

Abstract: The proposed mechanism for transcription coupled nucleotide excision repair (TCR) invokes RNA polymerase (RNAP) blocked at a DNA lesion as a signal to initiate repair. In Escherichia coli, TCR requires the interaction of RNAP with a transcription-repair coupling factor encoded by the mfd gene. The interaction between RNAP and Mfd depends upon amino acids 117, 118, and 119 of the β subunit of RNAP; changing any one of these to alanine diminishes the interaction . Using direct assays for TCR, and the lac operon of E. coli containing UV induced cyclobutane pyrimidine dimers (CPDs) as substrate, we have found that a change from arginine to cysteine at amino acid 529 of the β subunit of the RNAP inactivates TCR, but does not prevent the interaction of RNAP with Mfd. Our results suggest that this interaction may be necessary but not sufficient to facilitate TCR. [Copyright &y& Elsevier]