TRCF (Transcription Repair Coupling Factor) is a widely conserved bacterial protein that couples DNA repair with transcription. TRCF recognizes RNAP stalled at a non-coding template site of DNA damage, disrupts the transcription complex to release the transcript and RNAP, and recruits the DNA excision repair machinery to the site. The mechanism of RNA release has been illuminated by the discovery that TRCF causes forward translocation of RNAP, using an ATP-dependent motor that is highly homologous to that of the Holliday branch migration protein RecG. TRCF is a large (130 kDa), multi-functional protein with a complex structure/function relationship that is currently understood only from sequence analysis and genetic manipulation. In this grant, we propose detailed structural studies to elucidate the structure/function relationship of TRCF, to reveal conformational changes involved in the ATP-hydrolysis cycle and its coupling to the DNA translocase activity, and to reveal the interactions of TRCF with the RNAP ternary elongation complex. Specifically, we propose to: 1. Determine the X-ray crystal structure of TRCF. 2. Determine the structural basis for TRCF DNA translocase activity through X-ray crystal structures with nucleotides and/or nucleotide analogs. 3. Determine the structural basis for the specific transcription termination activity of TRCF through structural studies of an RNAP ternary elongation complex (RNAP/DNA/RNA) with TRCF.
| Howan, Kévin; Smith, Abigail J; Westblade, Lars F et al. (2012) Initiation of transcription-coupled repair characterized at single-molecule resolution. Nature 490:431-4 |
| Srivastava, Devendra B; Darst, Seth A (2011) Derepression of bacterial transcription-repair coupling factor is associated with a profound conformational change. J Mol Biol 406:275-84 |
| Opalka, Natacha; Brown, Jesse; Lane, William J et al. (2010) Complete structural model of Escherichia coli RNA polymerase from a hybrid approach. PLoS Biol 8: |
| Westblade, Lars F; Campbell, Elizabeth A; Pukhrambam, Chirangini et al. (2010) Structural basis for the bacterial transcription-repair coupling factor/RNA polymerase interaction. Nucleic Acids Res 38:8357-69 |
| Stallings, Christina L; Stephanou, Nicolas C; Chu, Linda et al. (2009) CarD is an essential regulator of rRNA transcription required for Mycobacterium tuberculosis persistence. Cell 138:146-59 |
| Deaconescu, Alexandra M; Savery, Nigel; Darst, Seth A (2007) The bacterial transcription repair coupling factor. Curr Opin Struct Biol 17:96-102 |
| Du, Yunrui; Walker, Lee; Novick, Peter et al. (2006) Ptc1p regulates cortical ER inheritance via Slt2p. EMBO J 25:4413-22 |
| Deaconescu, Alexandra M; Chambers, Anna L; Smith, Abigail J et al. (2006) Structural basis for bacterial transcription-coupled DNA repair. Cell 124:507-20 |
| Deaconescu, Alexandra M; Darst, Seth A (2005) Crystallization and preliminary structure determination of Escherichia coli Mfd, the transcription-repair coupling factor. Acta Crystallogr Sect F Struct Biol Cryst Commun 61:1062-4 |
