Lucia Rothman-Denes, Ph.D.

Bacterial and Bacteriophage Physiology and Transcription

Research Summary

Regulation of RNA polymerase activity is the main target of control of gene expression in all organisms. Our laboratory studies structure-function relationships, mechanisms of promoter recognition and mechanisms of activation of three DNA-dependent RNA polymerases (RNAP) using genetic, biochemical and biophysical approaches. Bacteriophage N4-coded virion RNAP (vRNAP) is a 3,500 amino acid long polypeptide that transcribes single-stranded, promoter containing templates with specificity. We have shown that determinants of promoter recognition include specific sequences and a small hairpin on the template strand, which is formed on double-stranded DNA through specific sequences and supercoiling. Promoter utilization requires E. coli single-stranded DNA binding protein (SSB) at physiological superhelical densities. The vRNAP domains responsible for promoter recognition and catalysis and the mechanism of Eco SSB activation are under investigation. Bacteriophage N4-coded RNAPII is a heterodimer with sequence homology to the T7 RNAP; however, it does not transcribe promoter-containing dsDNA templates. An additional small, phage-coded, which required in vivo for N4 RNAPII transcription, is a SSB that specifically recruits N4 RNAPII to ssDNA. The interaction of proteins at the promoter and the mechanism of promoter recognition are under investigation. The N4-coded SSB activates bacteriophage N4 late transcription through direct interaction with the ß' 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. Our studies have shown that, unexpectedly, single-stranded DNA binding proteins (SSB) act as transcription activators.

Selected Papers

Dai X, Greizerstein M, Nadas-Chinni K and Rothman-Denes LB. (1997). Supercoil-induced extrusion of a regulatory DNA hairpin, Proc. Natl. Acad. Sci. USA, 94, 2174-2179.

Miller A, Wood D, Ebright RH and Rothman-Denes LB. (1997). RNA polymerase ß' subunit: target of DNA-binding-independent transcriptional activation. Science, 275, 1655-1657.

Dai X and Rothman-Denes LB. (1998). Sequence and DNA structural determinants of N4 virion RNA polymerase-promoter recognition. Genes & Dev, 12, 2782-2790.

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

Rothman-Denes LB, 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

Dai X and Rothman-Denes LB. (1999). DNA Structure and Transcription. Current Op. Microbiol., Cell Regulation, Nikaido, H. and K. Chater eds. 2, 126-130

Kazmierczak KM, Davydova EK, Mustaev AA and Rothman-Denes LB. (2002). "The phage N4 virion RNA polymerase catalytic domain is related to single-subunit RNA polymerases." Embo J 21: 5815-5823. 

Willis SH, Kazmierczak KM, Carter RH and Rothman-Denes LB. (2002). "N4 RNA polymerase II, a heterodimeric RNA polymerase with homology to the single-subunit family of RNA polymerases." J Bacteriol 184: 4952-61. 

Carter RH, Demidenko AA, Hattingh-Willis S and Rothman-Denes LB. (2003). "Phage N4 RNA polymerase II recruitment to DNA by a single-stranded DNA-binding protein." Genes Dev 17: 2334-45. 

Davydova EK and Rothman-Denes LB. (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. 

Davydova EK, Kazmierczak KM and Rothman-Denes LB. (2003). "Bacteriophage N4-coded, virion-encapsulated DNA-dependent RNA polymerase." Methods Enzymol 370: 83-94.

Stojkevic E and Rothman-Denes LB.  (2007).  "Coliphage N4 N-Acetylmurimidase defines a new family of murein hydrolases."  J Mol Bio  366: 406-419.

Davydova EK, Santangelo T and Rothman-Denes LB.  (2007).  "Bacteriophage N4 virion RNA polymerase interaction with its promoter DNA hairpin."  Proc Nat Acad of Sci USA  104:  7033-7038.

Murakami K, Davydova EK and Rothman-Denes LB  (2008).  "X-ray crystal structure of the polymerase domain of bacteriophage N4 virion RNA polymerase."  Proc Nat Acad of Sci USA  105:  5036-5041.

Choi K, McPartland J, Kaganman I, Bowman V, Rothman-Denes LB and Rossmann M  (2008).  "Insights into DNA and protein transport in double-stranded DNA viruses:  The Structure of bacteriophage N4."  J Mol Bio  378:  726-736.