RNA polymerase structure/function; regulation of transcript elongation in bacteria and humans
Our
research focuses on structure/function of RNA polymerase (RNAP) and its
central role in the regulation of gene expression. The catalytic
activity of RNAP is highly regulated by signals in DNA and RNA and by
extrinsic regulators that determine whether or not genes and operons
are successfully transcribed. Befitting its importance in regulation,
RNAP is a complex enzyme with multiple subunits and many moving parts.
We seek to understand how different signals and regulators control RNAP
activity. We study this both at a basic structure/function level by
dissecting interactions and movements of the different parts of RNAP
and at a cellular level by determining how regulators affect RNAP?s
global distribution among and movement along genes and operons.
Current projects in the lab take distinctly different approaches,
ranging from genetics to biophysics, but all rely on each other for
overall progress.
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A paused transcription complex and regulatory inputs for bacterial RNAP, which consists of b', b, w, and two a
subunits. Nascent RNA hairpins can pause or terminate transcription.
NusA stimulates pausing and termination via contacts that include the
hairpin. Rho stimulates termination. NusG stimulates elongation. Human
RNAPII contains 12 instead of 5 subunits and is regulated by other
factors through related mechanisms. |
Regulation of Bacterial RNAP
Bacterial
RNAP is regulated by intrinsic RNA/DNA signals that cause pausing or
termination and by highly conserved elongation regulators such as Rho,
NusA, and NusG. Key goals of current research are to determine where
these regulators contact RNAP and how they alter its conformation. We
investigate these issues using molecular genetics, protein-protein
crosslinking and footprinting, rapid quench-flow kinetics, and protein
engineering. A current high priority is to obtain a crystal structure
of a paused transcription complex to learn how the active site is
rearranged in the paused state. We also use single-molecule
transcription assays with our ?tri-lab? collaborators at Stanford and
Brandeis, for which visualizing the effects of elongation regulators on
RNAP movement is a high priority.
Regulation of Human RNA Polymerase II
Control
of transcript elongation plays central roles in human biology, for
instance by regulating HIV-1 gene expression, controlling gene
expression during differentiation (e. g., conversion of stem
cells to specialized cells), and controlling mRNA splicing and
processing, which are coupled to transcript elongation. However, the
mechanisms by which elongation regulators control human RNAPII are
largely unknown. We are investigating human RNAPII elongation control
in several ways. First, we have developed rapid kinetic assays to
dissect the elongation and pausing mechanism. Second, we are using
site-directed mutagenesis to create specifically altered human RNAPII
enzymes to investigate these mechanisms as well as how elongation
regulators like DISF/NELF and TFIIS alter with RNAPII. Finally, we are
developing single-molecule assays for human RNAPII so that we can
exploit approaches similar to those we apply to bacterial RNAP.
Study of Transcriptional Regulation in vivo
We
study transcription in cells using two different approaches. In
collaboration with Aseem Ansari (Biochemistry) and Tricia Kiley
(Biomolecular Chemistry), we study the global distributions of RNAP and
regulators in microbial cells growing in different conditions using
so-called ChIP chip assays. These experiments promise to revolutionize
our understanding of transcriptional regulation by showing how RNAP and
regulators are distributed among genes as cells change their regulatory
programs. We also are developing methods to produce recombinant RNAPs
in E. coli from diverse bacteria, especially from
difficult-to-study pathogens. We use the recombinant RNAPs to study
their novel regulatory properties. This approach also is being
developed as a high-throughput whole-cell assay for novel and
lineage-specific inhibitors with potential applications as new
antibiotics.