This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Regulation of gene expression during infection of the thermophilic bacterium Thermus thermophilus (T. th.) HB8 with the bacteriophage P23-45 was investigated. Macroarray analysis revealed host transcription shut-off and identified three temporal classes of phage genes: early, middle, and late. Primer extension experiments revealed that the 5? ends of P23-45 early transcripts are preceded by a common sequence motif that likely defines early viral promoters. T. th. HB8 RNA polymerase (RNAP) recognizes middle and late phage promoters in vitro but does not recognize early promoters. In vivo experiments revealed the presence of rifampicin-resistant RNA polymerizing activity in infected cells responsible for early transcription. The product of the P23-45 early gene 64 shows a distant sequence similarity with the largest, catalytic subunits of multisubunit RNAPs and contains the conserved metal-binding motif that is diagnostic of these proteins. We hypothesize that ORF64 encodes rifampicin-resistant phage RNAP that recognizes early phage promoters. Affinity isolation of T. th. HB8 RNAP from P23-45-infected cells identified two phage-encoded proteins: gp39 and gp76, that bind the host RNAP and inhibit in vitro transcription from host promoters, but not from middle or late phage promoters, and may thus control the shift from host to viral gene expression during infection. To our knowledge, gp39 and gp76 are the first characterized bacterial RNAP-binding proteins encoded by a thermophilic phage.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000862-38
Application #
8361589
Study Section
Special Emphasis Panel (ZRG1-BCMB-Q (40))
Project Start
2011-03-01
Project End
2012-03-31
Budget Start
2011-03-01
Budget End
2012-03-31
Support Year
38
Fiscal Year
2011
Total Cost
$1,304
Indirect Cost
Name
Rockefeller University
Department
Miscellaneous
Type
Other Domestic Higher Education
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
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
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
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
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