The goal of this project is to understand the mechanism of transcription termination during eukaryotic mRNA synthesis. The problem is being approached in a viral model system, vaccinia, where biochemical and genetic complexity are reduced relative to that of the cell. Vaccinia early mRNAs are synthesized by a multisubunit virus-encoded RNA polymerase. Transcription of early genes terminates downstream of a cis- acting termination signal, UUUUUNU, in the nascent RNA. A separate factor, termed VTF (vaccina termination factor), is required for termination in vitro by purified vaccinia RNA polymerase. Extensive purification of VTF, and the reconstitution of VTF from its constituent polypeptides, has established that the termination factor is identical to the vaccinia mRNA capping enzyme, a virus-encoded protein of 95 kDa and 31 kDa subunits. This proposal outlines a combined biochemical and molecular genetic analysis of the role of the multifunctional capping enzyme in mRNA biogenesis, focusing on the following questions. How are distinct functional domains organized within the heterodimeric VTF/capping enzyme molecule? How does the factor interact with other constituents of the transcriptional apparatus? What is the structure of the polymerase elongation complex? What subunit interactions contribute to the assembly and function of the vaccinia RNA polymerase? To answer these questions, the vaccinia genes encoding the two subunits of VTF/capping enzyme have been expressed in bacteria. By assessing the effects of amino acid sequence alterations on enzyme activity in vitro, we will fine-map the three individual catalytic domains for RNA modification (triphosphatase, guanylyltransferase, and methyltransferase) and will delineate the (as yet) uncharted requirements for factor-dependent termination. Probing the structure of the ternary complex will entail purification of active complexes of RNA polymerase halted in vitro at unique template positions. UV crosslinking will be used to determine which polypeptide subunits of the elongation complex make contact with nascent RNA and with the DNA template. Analysis of transcription complexes halted at various intervals on the template will reveal whether, and how, the complex changes as polymerase moves away from the promoter and as the termination signal is transcribed. The """"""""two-hybrid system"""""""" in yeast will be exploited to define the spectrum of binary protein-protein interactions among the vaccinia gene products involved in early transcription. The proposed studies of viral transcription termination should provide insights into analogous aspects of cellular mRNA synthesis. Indeed, termination has been implicated as a regulatory step in the expression of cellular proto-oncogenes (e.g. c-myc) during tumorigenesis, and in the genetic program of human immunodeficiency virus (HIV).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM042498-06
Application #
2181423
Study Section
Experimental Virology Study Section (EVR)
Project Start
1989-07-01
Project End
1998-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
6
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Bacusmo, Jo Marie; Orsini, Silvia S; Hu, Jennifer et al. (2018) The t6A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in Streptococcus mutans. RNA Biol 15:508-517
Schmier, Brad J; Shuman, Stewart (2018) Deinococcus radiodurans HD-Pnk, a Nucleic Acid End-Healing Enzyme, Abets Resistance to Killing by Ionizing Radiation and Mitomycin C. J Bacteriol 200:
Remus, Barbara S; Goldgur, Yehuda; Shuman, Stewart (2017) Structural basis for the GTP specificity of the RNA kinase domain of fungal tRNA ligase. Nucleic Acids Res 45:12945-12953
Remus, Barbara S; Schwer, Beate; Shuman, Stewart (2016) Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis. RNA 22:1500-9
Unciuleac, Mihaela-Carmen; Goldgur, Yehuda; Shuman, Stewart (2015) Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase. Proc Natl Acad Sci U S A 112:13868-73
Unciuleac, Mihaela-Carmen; Shuman, Stewart (2015) Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi. RNA 21:824-32
Das, Ushati; Wang, Li Kai; Smith, Paul et al. (2014) Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP•Mg2+ and 5'-OH oligonucleotide and a product complex with GDP•Mg2+ and 5'-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoac Nucleic Acids Res 42:1152-61
Das, Ushati; Wang, Li Kai; Smith, Paul et al. (2014) Structures of bacterial polynucleotide kinase in a michaelis complex with nucleoside triphosphate (NTP)-Mg2+ and 5'-OH RNA and a mixed substrate-product complex with NTP-Mg2+ and a 5'-phosphorylated oligonucleotide. J Bacteriol 196:4285-92
Remus, Barbara S; Jacewicz, Agata; Shuman, Stewart (2014) Structure and mechanism of E. coli RNA 2',3'-cyclic phosphodiesterase. RNA 20:1697-705
Chakravarty, Anupam K; Smith, Paul; Jalan, Radhika et al. (2014) Structure, mechanism, and specificity of a eukaryal tRNA restriction enzyme involved in self-nonself discrimination. Cell Rep 7:339-347

Showing the most recent 10 out of 98 publications