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 SH (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 a-helical coiled-coil dimer which extends into solution. Crosslinking studies 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 and unusual asymmetric charge distribution. One face of the protein is highly acidic, whereas the opposite face is neutral except for small basic patch. These properties suggest a model of how GreA interacts with the elongating 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 relationship 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 mechanisms of the transcript cleavage reaction induced by GreA will shed light on the mechanism of RNA activity and elongation itself.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000862-25
Application #
6279522
Study Section
Project Start
1997-12-01
Project End
1998-11-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
25
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Type
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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
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
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
Xue, John Z; Woo, Eileen M; Postow, Lisa et al. (2013) Chromatin-bound Xenopus Dppa2 shapes the nucleus by locally inhibiting microtubule assembly. Dev Cell 27:47-59
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

Showing the most recent 10 out of 67 publications