Research Summary

1. RNAP structure, function and mechanisms of regulation:

We use genetic, biochemical and biophysical approaches to study three DNA-dependent RNA polymerases (RNAP). Phage N4-coded virion RNAP (vRNAP) is a 3,500 aa polypeptide composed of three domains: the NTD required for genome injection, the RNAP domain (the most evolutionary diverged member of the T7 RNA family), and the CTD required for RNAP encapsidation. vRNAP transcribes single-stranded DNAs containing a small hairpin and specific sequences with exquisite specificity. vRNAP requires the E. coli single-stranded DNA binding protein (SSB) for promoter activation and transcript elongation. The crystal structures of the RNAP domain and of the RNAP-promoter complex (in collaboration with Dr K. Murakami, Penn State) reveal the mechanism of vRNAP hairpin promoter recognition and its exquisite specificity. The determinants of vRNAP-SSB interaction are under investigation. Bacteriophage N4-coded RNAPII is a heterodimer with sequence homology to the T7 RNAP, but does not transcribe promoter-containing dsDNA templates. It requires a small, phage-coded protein (gp2, SSB), which specifically recruits N4 RNAPII to ssDNA. The interaction of proteins at the promoter, and the mechanism of promoter recognition are under study. The N4-coded SSB activates N4 late transcription through direct interaction with the ' subunit of E. coli RNAP, i.e. acting as an allosteric effector. The mechanism of activation and the role of this region of RNAP in E. coli transcription are being studied.

2. Host-phage interactions:

We investigate the mechanism and regulation of N4-induced cell lysis, the mechanisms of N4-induced shut off host DNA replication and septation by two small N4-coded proteins. The recent elucidation of the structure of N4 virions in collaboration with Drs K. Choi and M. Rossmann (Purdue University) provides insights into the mechanism of N4 attachment and vRNAP and genome injection into the host, which are under study.

Selected Publications

Strojkovic, E. and Rothman-Denes, L.B. (2007) "Coliphage N4 N-acetylmuraminidase defines a new family of murein hydrolases" J Mol Biol 366: 406-419. (PubMed)

Carter, R. H., Demidenko, A. A., Hattingh-Willis, S. and Rothman-Denes, L. B. (2003). "Phage N4 RNA polymerase II recruitment to DNA by a single-stranded DNA-binding protein." Genes Dev 17: 2334-45. (PubMed)

Davydova, E. K. and Rothman-Denes, L. B. (2003). "Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase." Proc Natl Acad Sci U S A 100: 9250-5. (PubMed)

Davydova, E. K., Kazmierczak, K. M. and Rothman-Denes, L. B. (2003). "Bacteriophage N4-coded, virion-encapsulated DNA-dependent RNA polymerase." Methods Enzymol 370: 83-94. (PubMed)

Kazmierczak, K. M., Davydova, E. K., Mustaev, A. A. and Rothman-Denes, L. B. (2002). "The phage N4 virion RNA polymerase catalytic domain is related to single-subunit RNA polymerases." Embo J 21: 5815-5823. (PubMed)

Rothman-Denes, L. B., Dai, X., Davydova, E., Carter, R., and Kazmierczak, K. 1999. Transcriptional Regulation by DNA Structural Transitions and Single-Stranded DNA Binding Proteins. 63rd Cold Spring Harbor Symp. Quant. Biol. 63:63-73 (PubMed)

Dai, X. and Rothman-Denes, L. B. (1998). "Sequence and DNA structural determinants of N4 virion RNA polymerase-promoter recognition." Genes Dev 12: 2782-90. (PubMed)

Dai, X., Kloster, M. and Rothman-Denes, L. B. (1998). "Sequence-dependent extrusion of a small DNA hairpin at the N4 virion RNA polymerase promoters." J Mol Biol 283: 43-58. (PubMed)