The proposed research represents our longstanding efforts to understand the structure, mechanisms of promoter recognition, polymerase and promoter activation, and transcription initiation and elongation of two phage N4-coded RNA polymerases (RNAP), vRNAP and N4 RNAPII. These RNAPs belong to the T7-like "single-subunit" RNAP family that includes mitochondrial RNAPs. In contrast to the archetypal T7 RNAP, the N4-encoded and mitochondrial RNAPs require protein factors throughout the transcription cycle. We recently reported the X-ray crystal structures of the N4 mini-vRNAP, the catalytic component of vRNAP, in the apo-form and hairpin-form promoter bound and transcription initiation complexes. This represents a major breakthrough and provides the foundation for addressing questions on the transcription mechanism of factor-dependent single-subunit RNAPs using multidisciplinary approaches as described below. 1. Functional and structural dissection of the vRNAP polypeptide: The vRNAP apo-form is in an inactive conformation, and undergoes a structural rearrangement that leads to activation upon promoter binding. We will identify the "sensor" in vRNAP and the "signal" in the hairpin-form promoter that lead to vRNAP activation using biochemical approaches. In addition, we will solve the structure of mini-vRNAP in the transition from apo- form to binary complex by improving preliminary crystals that diffract to 3.4 ? resolution. Mini-vRNAP uses a transcription elongation factor, EcoSSB, to displace the RNA product from the DNA template. We will identify the EcoSSB site of interaction in the transcription elongation complex (TEC) using site-specific protein-protein crosslinking followed by site-directed mutagenesis. In contrast to T7 RNAP, mini-vRNAP is refractory to loading the DNA/RNA scaffold for TEC formation. Therefore, we will use structure-based protein engineering and the unique heat plasticity of vRNAP to prepare the mini-vRNAP TEC with DNA/RNA scaffold, and solve its structure with or without a EcoSSB peptide. 2. Elucidate the N4 RNAPII mechanism of promoter recognition: N4 RNAPII uses a transcription factor N4gp2, a single-stranded DNA binding protein, to recognize its promoters, which are similar in position and length to yeast mitochondrial RNAP promoters. Using an in vivo reporter assay, we will identify the N4 RNAPII determinants of promoter recognition by isolating N4 RNAPII derivatives that suppress promoter mutations. We will define the gp2 interaction platform on N4 RNAPII by site-specific protein-protein crosslinking followed by site-directed mutagenesis. We will also probe the role of gp2 in N4 RNAPII transcription elongation. To obtain a structural basis for factor-dependent single-subunit RNAP transcription, we will solve the crystal structures of the N4 RNAPII apo-form, promoter bound and TEC with or without gp2. We have recently identified and additional protein, N4gp1, required for N4 RNAPII transcription. We will determine its localization on the template in vivo, purify gp1 and analyze its effect on N4 RNAP transcription initiation.

Public Health Relevance

The transcription process by bacteriophage N4-coded RNA polymerases has intriguing similarity to that found in mitochondria. Therefore, our multidisciplinary study of N4 RNA polymerases and transcription will provide a framework for understanding gene expression in mitochondria that generates over 90 % of the energy used by mammalian cells and also plays a key role in cell death signaling pathways and numerous disease states (Alzheimer's and Parkinson's diseases, osteoarthritis, type 2 diabetes mellitus, and cancer).

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI012575-37
Application #
8291346
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Korpela, Jukka K
Project Start
1988-05-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
37
Fiscal Year
2012
Total Cost
$579,320
Indirect Cost
$146,227
Name
University of Chicago
Department
Genetics
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Basu, Ritwika S; Murakami, Katsuhiko S (2013) Watching the bacteriophage N4 RNA polymerase transcription by time-dependent soak-trigger-freeze X-ray crystallography. J Biol Chem 288:3305-11
Molodtsov, Vadim; Nawarathne, Irosha N; Scharf, Nathan T et al. (2013) X-ray crystal structures of the Escherichia coli RNA polymerase in complex with benzoxazinorifamycins. J Med Chem 56:4758-63
Chen, Yuanyuan; Basu, Ritwika; Gleghorn, Michael L et al. (2011) Time-resolved events on the reaction pathway of transcript initiation by a single-subunit RNA polymerase: Raman crystallographic evidence. J Am Chem Soc 133:12544-55
Yano, Sho T; Rothman-Denes, Lucia B (2011) A phage-encoded inhibitor of Escherichia coli DNA replication targets the DNA polymerase clamp loader. Mol Microbiol 79:1325-38
Gleghorn, Michael L; Davydova, Elena K; Basu, Ritwika et al. (2011) X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides. Proc Natl Acad Sci U S A 108:3566-71
Davydova, Elena K; Kaganman, Irene; Kazmierczak, Krystyna M et al. (2009) Identification of bacteriophage N4 virion RNA polymerase-nucleic acid interactions in transcription complexes. J Biol Chem 284:1962-70
McPartland, Jennifer; Rothman-Denes, Lucia B (2009) The tail sheath of bacteriophage N4 interacts with the Escherichia coli receptor. J Bacteriol 191:525-32
Gleghorn, Michael L; Davydova, Elena K; Rothman-Denes, Lucia B et al. (2008) Structural basis for DNA-hairpin promoter recognition by the bacteriophage N4 virion RNA polymerase. Mol Cell 32:707-17
Murakami, Katsuhiko S; Davydova, Elena K; Rothman-Denes, Lucia B (2008) X-ray crystal structure of the polymerase domain of the bacteriophage N4 virion RNA polymerase. Proc Natl Acad Sci U S A 105:5046-51
Choi, Kyung H; McPartland, Jennifer; Kaganman, Irene et al. (2008) Insight into DNA and protein transport in double-stranded DNA viruses: the structure of bacteriophage N4. J Mol Biol 378:726-36

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