We strive to understand the physical mechanisms of transcription initiation and other important DNA-protein interactions. More specifically, we use a variety of single-molecule and bulk biophysical techniques including both optical and magnetic tweezers and fluorescent microscopy to investigate how the assembly of initiation complexes on DNA promoters lead to transcription of genes. Our work is currently focused on the mechanisms of basal transcription initiation in Eukaryotes and on factor-regulated transcription in Mycobacterium tuberculosis.
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Mechanisms of the eukaryotic pre-initiation complex
The ability of cells to interact and respond to their environment is largely dependent on the regulation of gene expression. Much of this regulation occurs at the stage of transcription initiation where an RNA polymerase is recruited to a promoter, the DNA is unwound, and the polymerase begins processive transcription elongation. In Eukaryotes, basal and activated initiation is dependent on the formation of a “pre-initiation complex” or PIC that is made up of over 30 polypeptide chains and contains a variety of DNA binding and catalytic activities (EMD-8132 and PDB-5IY7). For example, as many as three different ATPase centers collaborate to produce an elongating polymerase. We are interested in dissecting the molecular mechanism of the PIC to set the stage for a deeper understanding of how this process is regulated during changes in gene expression as well as how the process may go awry in certain disease states.
Transcription Start Site Scanning and the Requirement for ATP During Transcription Initiation by RNA Polymerase II.
Fishburn J, Galburt EA, Hahn S
Journal of Biological Chemistry. 2016 April; 291:13040-13047.
Double-stranded DNA translocase activity of TFIIH and the mechanism of RNA Polymerase II Open Complex formation.
Fishburn J, Tomko EJ, Galburt EA, Hahn S
PNAS. 2015 March; 112(13):3961-6.
Unique and essential transcription factors in Mycobacterium tuberculosis
Approximately one-third of the world population is infected with Mycobacterium tuberculosis and millions die from this disease annually. The success of this pathogen lies in its ability to lay dormant for long periods of time and to resist the attempts of the immune system to eradicate it from the host. Although much is known about transcription initiation in model bacterial such as Escherichia coli, relatively little is known about the mechanisms of basal transcription in mycobacteria. For example, transcription factors that are essential in M. tuberculosis such as CarD and RbpA are not even present in the genome of E. coli. These factors bind directly to the RNA polymerase as shown to the left (structures 4xls and 5tw1). We are studying the mechanisms of transcription initiation in this important pathogen to better understand the diversity of gene expression pathways in bacteria. In addition, as multi-drug resistant strains are becoming more prevalent we hope to find novel molecular strategies to interfere with the unique regulatory pathways of M. tuberculosis and treat this costly disease.
Cooperative stabilization of Mycobacterium tuberculosis rrnAP3 open promoter complexes by RbpA and CarD.
Rammohan J, Ruiz Manzano A, Garner AL, Prusa J, Stallings CL, Galburt EA
Nucleic Acids Research. 2016 June; 44(15):7304-7313.
CarD stabilizes mycobacterial open complexes via a two-tiered kinetic mechnansim.
Rammohan J, Ruiz Manzano A, Garner AL, Stallings CL, Galburt EA
Nucleic Acids Research. 2015 February; 43(6):3272-85.