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Research Overview

Our research focuses on
(1) RNA polymerase, the central enzyme of gene expression in all free-living organisms;
(2) the mechanisms by which gene expression by RNA polymerase is regulated and can be re-programmed for biodesign; and
(3) applications of these basic research advances to microbial biotechnology and to antibiotic discovery.

Our basic research focus is to understand how the fundamental properties of RNA polymerase, largely conserved from bacteria to human, make it susceptible to pausing, arrest, or termination and how elongation regulators, nucleoprotein structures, and metabolic, developmental, and environmental signals alter these properties. We use a variety of approaches, including genetics, biomolecular chemistry, synthetic biology, systems biology, biophysics, and structural biology, to study both fundamental and applied paradigms of gene regulation.



Projects

  1. Transcription Complex Structure/Function
  2. Transcriptional Dynamics of Bacterial Chromosomes
  3. Microbial Biodesign / Synthetic Regulatory Circuits
  4. Transcription Termination Mechanisms
  5. Diverse RNA Polymerases / Antibiotic Discovery
  6. Transcription by Single RNA Polymerase Molecules



1. Transcription Complex Structure/Function     back to top

RNA polymerases respond to intrinsic signals in DNA and RNA that cause the enyzmes to pause, arrest, or terminate RNA synthesis. Many regulatory molecules function by overriding or enhancing these intrinsic signals. To understand the fundamental mechanisms of these intrinsic signals, we dissected pause signals because they (i) play key roles in gene regulation by synchronizing movements of RNA polymerase on genes with binding of regulatory factors to RNA polymerase and nascent RNA, (ii) couple transcription and translation in bacteria, (iii) facilitate proper RNA folding, and (iv) are precursors to arrest and termination. We found that pause signals come in at least two classes: those stabilized by RNA hairpins, and those stabilized by backtracking of RNA polymerase on the RNA and DNA. Both classes appear to be generated from a common elemental pause intermediate that arises by a rearrangement in RNA polymerase's active site.

Two classes of pause signals. Pausing arises when nucleic acid interactions in the elongation complex lead to a loosening of the clamp domain and incomplete translocation, trapping RNA polymerase in the elemental paused state. The hairpin-stabilized pause is generated from an elemental pause when the nascent RNA folds into "pause hairpin" that jams open the clamp domain and traps the trigger loop in an inactive conformation. (click here to see figure of the overall pause mechanism)

Current research foci are:

  • What conformational changes in RNA polymerase underlie formation of the elemental paused complex?
  • How do pause hairpins stabilize the paused conformation?
  • What is the detailed kinetic mechanism of transcriptional pausing?
  • What are the kinetics of RNA hairpin formation relative to the events of pausing and hairpin-dependent termination?

Relevant publications:




2. Transcriptional Dynamics of Bacterial Chromosomes     back to top

In eukaryotes, histones can silence specific genes through the formation of heterochromatin. In bacteria, gene silencing occurs by a mechanism that is largely unknown but is essential for regulation of horizontally acquired genes. A variety of proteins, generally known as nucleoid-associated proteins (NAPs), play roles in both compacting the genome and in silencing various genes. Many details about how these proteins affect the nucleoid both individually and in combination are unknown. Our lab is working to answer questions about how these NAPs regulate transcription by RNA polymerase through in vivo and in vitro techniques. The NAPs we are interested in are a part of the H-NS family of proteins and include H-NS, its paralog StpA and two closely related paralogs, Hha and YdgT. Previously, our lab found that H-NS synergizes with Rho which suppresses a vast amount of anti-sense transcription. We are still investigating mechanisms by which this can happen.

Effects of H-NS family of NAPs on elongating RNA polymerase. The H-NS family of NAPs can bind to DNA in several combinations to silence DNA. Here H-NS is shown in a bridged (red) or linear (purple) filament. This filament could include StpA (gray), Hha (brown), or YdgT (blue), each of which likely modifies the overall structure of the filament. A homogenous StpA filament could also exist (gray). How these different filament structures affect RNA polymerase (light blue oval) is still unknown.

Current research foci are:

  • What are the effects of histone-like proteins (Hu, H-NS, Fis) in E. coli on transcription elongation?
  • How do elongation factors affect the response of RNA polymerase to histone-like proteins?
  • What are the dynamics of the H-NS filaments both in vitro and in vivo?
  • What effect do the other H-NS family proteins (StpA, Hha, and YdgT) have on an elongating RNA polymerase?

Relevant publications:



3. Microbial Biodesign / Synthetic Regulatory Circuits     back to top

The conversion of hydrolysates to biofuels requires organisms to tackle two problems: converting energy rich carbon sources to usable fuel compounds, and resisting toxicity caused by the variety of compounds generated during biomass liquefaction and hydrolysis. Our lab is focusing on mechanisms of stresses exerted on E. coli and S. cerevisiae by so-called lignotoxins alone and in combination with biofuel endproducts like ethanol. We are developing in vivo methods to accelerate metabolic engineering of microbial strains. Our goal is to create a synergy between in silico and in vivo approaches to generate the optimal patterns of gene expression so as to maximize the production of desired products from concentrated lignocellulosic hydrolysates or other renewable carbon sources.

Feedback control for microbial biodesign

Current research foci are:

  • Feedback control of ethanologenesis in E. coli
  • Sensing and detoxification of inhibitors in lignocellulosic hydrolysates
  • Generate strategies for optimization of native expression feedback controls

Relevant publications:



4. Transcription Termination Mechanisms     back to top

The remarkable stability of the transcription elongation complex is a major contributor to the enzyme's incredible processivity, but the difficulty in breaking the contacts between RNA polymerase and the DNA/RNA scaffold poses a significant barrier to transcription termination. To overcome this barrier, bacteria have evolved two major termination pathways: (i) intrinsic termination, wherein the nascent RNA folds into a hairpin that embeds itself in the RNA polymerase RNA exit channel, disrupting the transcription elongation complex, and (ii) Rho-dependent termination, which is mediated by the RNA helicase Rho. While these two pathways are generally understood, several of the mechanistic details remain poorly characterized. To address these questions, we have identified antisense transcription as a major site of Rho activity, reinforcing its role as the transcriptional housekeeper, and are continuing to investigate the kinetic parameters of both termination pathways.

Transcription Termination

Pathways to termination. (a) During intrinsic termination, RNA polymerase pauses at a U-rich tract, opening a kinetic window during which a GC-rich hairpin can nucleate in the RNA exit channel, dissociating the elongation complex. (b) Rho factor binds C-rich RNA sequences and hydrolyses ATP to translocate towards the elongation complex. At the moment of collision, Rho induces dissociation of the elongation complex through a poorly characterized mechanism.

Current research foci are:

  • What conformation changes must RNA polymerase undergo during termination?
  • What are the rate-limiting steps during intrinsic termination?
  • What are the binding determinants for Rho association with the elongation complex?
  • How do the general transcription factors NusG and NusA modulate Rho-dependent termination?
  • How do nucleoid-associate proteins, such as NAPs, interact with the elongation complex and Rho to facilitate termination?

Relevant publications:



5. Diverse RNA Polymerases/Antibiotic Discovery     back to top

We know a great deal about E. coli RNA polymerase as well as some eukaryotic RNA polymerases but we know surprisingly little about the diverse RNA polymerases in different bacterial species, other than that their properties may differ significantly from E. coli RNA polymerase. With the advent of routine genomic sequencing of diverse bacterial species and the development of methods to overproduce multisubunit RNA polymerases in E. coli, it is now possible to overexpress and purify some of these interesting enzymes without the need to handle the potentially hazardous organisms. We are now engaged in such efforts, focused currently on the RNA polymerases from M. tuberculosis and B. anthracis (the causative agents of tuberculosis and anthrax, respectively). Not only will our study of these enzymes yield important new information on the diversity of regulatory mechanisms for diverse bacteria, but they also will provide important tools for screening for new inhibitors on RNA polymerases from bacterial pathogens. These inhibitors can lead to insights into compounds for antibiotic development and the mechanism of transcription by RNA polymerase. Efforts to date have focused on two classes of novel RNA polymerase inhibitors: microcin J25 in collaboration with the labs of Seth Darst at Rockefeller University and Konstantin Severinov at Rutgers University, and the CBR class of inhibitors studied in collaboration with Cumbre, Inc. of Dallas, TX.

Current research foci are:

  • Overexpress and purify RNA polymerases from M. tuberculosis and B. anthracis.
  • Determine the response of novel RNA polymerases to intrinsic regulatory signals.
  • Identify novel RNA polymerases from genomic sequence information for future study.
  • Develop new screening methods to identify inhibitors of RNA polymerases from bacterial pathogens.
  • Obtain a crystal structure of the CBR class of inhibitor bound to RNA polymerase.

Relevant publications:



6. Transcription by Single RNA Polymerase Molecules     back to top

In 1991, we pioneered the study of single RNA polymerase molecules by developing a method to detect movement of RNA polymerase along a DNA template using optimal microscopy. The initial, albeit crude, technique opened the door to a rapidly growing field of study. Our single molecule project is now a mature three-way collaboration with the labs of Steve Block at Stanford University and Jeff Gelles at Brandeis University. Over the past couple of decades, the collaboration has yielded important new insights into the mechanism of transcription, culminating in publications showing that formation of the unactivated intermediate does not involve a change in the translocation state of RNA polymerase and detection of backtracking movements by RNA polymerase.

Laser trap used to hold a single RNA polymerase molecule during transcription. A streptavidin-coated bead is attached to an RNA polymerase that contains biotin at the C-terminus of the beta' subunit. One end of the DNA is attached to the microscope stage via a digoxigenin-antibody linkage. A constant force is maintained on the bead in the laser trap by feedback adjustment of the stage position as RNA polymerase transcribes. In this arrangement, assisting force is applied to the RNA polymerase. By instead locating the digoxigenin tag at the other end of the DNA, an opposing force to transcription can be created.

Current research foci are:

  • Detect and characterize pausing at defined pause signals by single molecule methods.
  • Characterize the effects of transcription factors NusA, NusG, and GreB on transcription by single RNA polymerase molecules.

Relevant publications: