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Associate Professor, Biochemistry Associate Professor, Fels Institute for Cancer Research and Molecular Biology Chairperson, Graduate Admissions Committee Telephone: 215-707-8152 Fax: 215-707-7536 Email: stitt@temple.edu
Department of BiochemistryFels Institute for Cancer Research and Molecular Biology
Transcription termination; transduction of ATP hydrolysis energy to directional movement; protein-RNA interactions.
Mechanism of E. coli transcription termination factor Rho Gene expression at the level of mRNA production is regulated at both the initiation and the termination of transcription. Transcription termination factor Rho is a homohexameric protein that releases newly synthesized RNA from Escherichia coli transcription complexes. Rho binds to a growing RNA chain and acts through ATP-fueled, 5' -> 3' travel along the nascent RNA. This directional movement is achieved by coordination of an RNA-dependent ATPase activity of Rho with the binding and release of RNA from the protein. When Rho catches up with the transcribing RNA polymerase, continued travel by Rho could unwind the RNA/DNA helix of the transcription bubble (helicase activity), which would complete disruption of the transcription complex.
Our goal is to understand the mechanism of directional travel by Rho along RNA, the basis for its helicase function. Despite having six identical subunits, Rho behaves like a trimer of dimers. We have found that Rho binds three molecules of ATP per hexamer, and hydrolyzes them sequentially upon RNA binding.
Rho can bind an 80-base length of RNA and protect it from ribonuclease degradation. The large size of the protected RNA suggests the presence of multiple RNA binding sites on the Rho hexamer, and two classes of sites have been identified.
We use molecular biological and enzymological approaches to understand Rho and its coordination of ATP hydrolysis with RNA binding and release. For example, site-specific mutagenesis is used to generate proteins whose behaviors are analyzed by steady-state and pre-steady-state kinetic measurements of ATP hydrolysis and by gel electrophoresis of in vitro transcription termination products.
Current work focuses on defining the roles of putative ATPase active site residues, on understanding protein conformation changes during catalysis, and on identifying possible sites of interaction on Rho with molecules of the transcription complex such as RNA polymerase and NusG.
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barbara L. Stitt, PhD