Our overall goal is to elucidate how the process of transcription initiation and activation is regulated at a molecular level.
Controlling the process of transcription is fundamental to gene expression, gene regulation, and development. In all organisms, RNA polymerase, a complex protein machine that transcribes genomic DNA into RNA, performs transcription. A single RNA polymerase in bacteria and archaea—and only three different polymerases in eukaryotes—program an amazing variety of developmental pathways.In most cases, factors that alter the initiation, elongation, or termination of transcription control the vast array of transcriptional outcomes.
Because transcription initiation proceeds through multiple steps, this process provides multiple points for regulation. In particular, transcriptional activators, co-activators, and repressors can interact with the template DNA and/or RNA polymerase to modulate core promoter selection. Early work suggested that the process and regulation of transcription initiation fundamentally differed between bacterial polymerase and higher organisms. However, recent work demonstrates that all kingdoms of life retain many features of this process. Recent biochemical and structural studies reveal significant functional similarities among bacterial, archaeal, and eukaryotic RNA polymerases. Throughout life, factors that interact with RNA polymerase and with sequences close to or within the core promoter itself can alter promoter recognition. In many cases, these factors interact with only a small surface of RNA polymerase, yet they impose a major specificity change through this contact.
My lab focuses on elucidating these mechanisms of transcription initiation and regulation. We employ simple bacterial and bacteriophage model systems, because these systems can be defined in detail biochemically and investigated at a molecular level. In particular, we have investigated the activation of phage T4 promoters during T4 infection and the regulation of promoters that express virulence gene products in the human pathogen Bordetella pertussis.
Our work on T4 promoter activation established a new paradigm for transcriptional activation, called sigma appropriation. We demonstrated how the binding of a small T4 protein structurally remodels a portion of the specificity subunit (sigma) of RNA polymerase. This, in turn, allows a T4 activator to interact with a portion of sigma that would normally be occluded by RNA polymerase and to interact with a portion of the DNA that would normally be bound by sigma. These interactions result in the formation of a remodeled specificity factor for RNA polymerase that recognizes a new promoter sequence. This work reveals at a molecular level how reconfiguring a small portion of RNA polymerase can completely alter promoter specificity.
To conduct our work on virulence gene regulation, we collaborate with the laboratory of Dr. Scott Stibitz at the U.S. Food and Drug Administration. B. pertussis is a reemerging pathogen and although there is a vaccine for pertussis, its effectiveness wanes after only a few years. We are interested in elucidating the details of gene regulation by the B. pertussis global response regulator, BvgA, which regulates all the known pertussis virulence genes. Our overall goal is to provide the intellectual basis for developing more effective strategies for combating the disease.Our work has revealed a unique architecture for the interaction of the Bordetella pertussis response regulator (BvgA) with a particular virulence gene promoter. We have found that BvgA and two subunits of RNA polymerase occupy the same region of DNA. Using molecular modeling, we have demonstrated how this is possible: they are located like spokes on a wheel around the DNA double-helix.
In both of our systems, we combine classic protein and nucleic acids biochemistry with state-of-the-art structural and molecular modeling techniques to understand the protein-protein and protein-DNA contacts that are needed for regulation.
Applying our Research
The emergence of antibiotic resistant pathogens forces us to consider other ways to combat bacterial infections. Our work contributes to the development of anti-microbial strategies using two systems. First, we investigate bacteriophages, viruses that infect bacteria and contain multiple mechanisms to disturb, overtake, and kill a bacterial cell. Bacteriophage T4 disrupts the ability of E. coli RNA polymerase to interact correctly with DNA. Our study of the molecular mechanism of this process yields insight into how small molecules can be generated to thwart bacteria. Second, we study Bordetella pertussis, which causes pertussis (whooping cough), a highly contagious upper respiratory infection. Although B. pertussis infections are one of the most prevalent preventable diseases, vaccine efficacy wanes after a few years; the recent increase in pertussis disease, including 27,550 cases in the United States in 2012, has been attributed in part to decreased vaccination coverage and suboptimal vaccines. Sadly, this has resulted in regional epidemics of pertussis and infant mortality. Understanding how B. pertussis expresses its virulence genes at a molecular level provides a basis for better drug and vaccine design.
Our work also has broader implications. There are multiple human diseases in which normal cell development has gone awry, such as cancer and autoimmune conditions like diabetes, rheumatoid arthritis, and Crohn’s disease. Although regulation of gene expression in human cells is certainly more complicated than that in bacteria, it is now clear that the basic steps of transcription initiation and activation are in fact shared among all organisms. Consequently, fundamental research into the mechanics of transcription and gene expression in simple bacterial systems provides insights that can be extended throughout biology.
Need for Further Study
The ability of RNA polymerase to correctly select the right promoter sequence at the right time is fundamental to the control of gene expression in all organisms. However, how the interaction of a factor or factors with RNA polymerase redirects polymerase to different promoter sequences is not well understood at a molecular level. This need for more knowledge exists despite available structures of RNA polymerases and various activators. A major question in our field is how contact between a small patch of protein with a small region of DNA or another protein can determine whether a gene is expressed or is silent.