Transcription elongation factors stimulate the activity of RNA polymerases (RNAPs) by increasing the overall elongation rate and the completion of RNA chains. One group of such factors, which includes E. coli GreA, GreB, and eukaryotic SII(TFIIS), acts by inducing hydrolytic cleavage of the transcript within the RNAP, followed by release of the 3'-terminal fragment. We have solved the structure of E. coli GreA to a resolution of 2.2 A. The structure contains a very unusual N-terminal domain consisting of an antiparallel alpha-helical coiled-coil dimer which extends into the solution. Crosslinking studes show that a site near the tip of the coiled-coil 'finger' plays a direct role in the transcript cleavage reaction by contacting the 3'-end of the transcript. The structure exhibits an unusual asymmetric charge distribution. One face of the protein is highly acidic, whereas the opposite face is neutral except for a small basic patch. These properties suggest a model of how GreA interacts with the elongation RNAP complex. It is not possible at this point to propose a more detailed model of how GreA induces transcript cleavage, but the structure now allows testing of specific elements by biochemical and genetic means. Guided by the X-ray crystal structure of GreA, we are using site-directed mutagenesis to dissect the structure/ function releationship of GreA. Once the mutant proteins are prepared, we depend on mass spectrometry to determine that we have the correct mutant, and that it is free of native GreA. We hope that elucidating the mechanism of the transcript cleavage reaction induced by GreA will shed light on the mechanism of RNAP activity and elongation itself.
| Manning, Lois R; Popowicz, Anthony M; Padovan, Julio C et al. (2017) Gel filtration of dilute human embryonic hemoglobins reveals basis for their increased oxygen binding. Anal Biochem 519:38-41 |
| Boice, Michael; Salloum, Darin; Mourcin, Frederic et al. (2016) Loss of the HVEM Tumor Suppressor in Lymphoma and Restoration by Modified CAR-T Cells. Cell 167:405-418.e13 |
| Chait, Brian T; Cadene, Martine; Olinares, Paul Dominic et al. (2016) Revealing Higher Order Protein Structure Using Mass Spectrometry. J Am Soc Mass Spectrom 27:952-65 |
| Krutchinsky, Andrew N; Padovan, Júlio C; Cohen, Herbert et al. (2015) Maximizing ion transmission from atmospheric pressure into the vacuum of mass spectrometers with a novel electrospray interface. J Am Soc Mass Spectrom 26:649-58 |
| Mast, Fred D; Rachubinski, Richard A; Aitchison, John D (2015) Signaling dynamics and peroxisomes. Curr Opin Cell Biol 35:131-6 |
| Krutchinsky, Andrew N; Padovan, Júlio C; Cohen, Herbert et al. (2015) Optimizing electrospray interfaces using slowly diverging conical duct (ConDuct) electrodes. J Am Soc Mass Spectrom 26:659-67 |
| Oricchio, Elisa; Papapetrou, Eirini P; Lafaille, Fabien et al. (2014) A cell engineering strategy to enhance the safety of stem cell therapies. Cell Rep 8:1677-1685 |
| Zhong, Yu; Morris, Deanna H; Jin, Lin et al. (2014) Nrbf2 protein suppresses autophagy by modulating Atg14L protein-containing Beclin 1-Vps34 complex architecture and reducing intracellular phosphatidylinositol-3 phosphate levels. J Biol Chem 289:26021-37 |
| Indiani, Chiara; O'Donnell, Mike (2013) A proposal: Source of single strand DNA that elicits the SOS response. Front Biosci (Landmark Ed) 18:312-23 |
| Di Virgilio, Michela; Callen, Elsa; Yamane, Arito et al. (2013) Rif1 prevents resection of DNA breaks and promotes immunoglobulin class switching. Science 339:711-5 |
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