Dept. of Microbiology and
Harvard Medical School
HIM Room 1027
4 Blackfan Circle
Boston, MA 02115
Transcription by the multi-subunit RNA polymerases is a complex process consisting of multiple steps at which regulation can occur. To understand the relevant molecular interactions and regulatory mechanisms, we are taking advantage of the relative simplicity of the prokaryotic transcription machinery and the power of bacterial genetics. The RNA polymerase (RNAP) core enzyme is evolutionarily conserved from bacteria to man, as has been strikingly confirmed by recent crystallographic studies. Accordingly, insights into the function of the bacterial enzyme are likely to be relevant to the function of all multi-subunit RNAPs. Because most regulation of gene expression occurs at the level of transcription, understanding how gene expression is misregulated in various disease states depends on a detailed understanding of RNAP function. In addition, RNAP remains an important antibiotic target and therefore mechanistic studies of RNAP will guide both the improvement of existing antibiotics and the development of new antibiotics that target RNAP.
We have developed a variety of genetic tools for examining functionally relevant protein-protein interactions between the subunits of RNAP, between transcription regulators and RNAP, and between the DNA-binding domains of RNAP and conserved promoter elements. Using these tools, we are probing the events that occur during transcription initiation and elongation and the way regulators modulate these events. Previously we have defined the minimal mechanistic requirements for the process of transcription activation by demonstrating that any sufficiently strong contact between a DNA-bound protein and a protein domain fused to RNAP can elicit transcription activation. Our findings provided the basis for establishing a prokaryotic counterpart to the familiar yeast-based two-hybrid assay for detecting protein-protein interactions in vivo. This bacterial two-hybrid assay under- lies many of the genetic strategies we are currently utilizing to dissect the transcription apparatus.
Our study of the protein-protein interactions that underlie transcription and its regulation has led to a more general interest in protein-protein interactions. A new project in the lab is focused on the potentially pathogenic protein-protein interactions that mediate the formation of prion-like protein aggregates. We are using E. coli cells to develop assays that would allow us to study prion proteins from other organisms and potentially to identify prion-like proteins of bacterial origin.
Dove S.L., Joung J.K., and Hochschild A. 1997. Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature 386: 627-630.
Gregory B. D., Nickels B. E., Garrity S. J., Severinova E., Minakhin L., Bieber-Urbauer R. J., Urbauer J. L., Heyduk T., Severinov K., and Hochschild A. 2004. A regulator that inhibits transcription by targeting an intersubunit interaction of RNA polymerase holoenzyme. Proc. Natl. Acad. Sci. USA 101: 4554-4559.
Jain D, Nickels BE, Sun L, Hochschild A, and Darst SA. 2004. Structure of a ternary transcription activation complex. Mol. Cell 13: 45-53.
Nickels, B.E., Garrity S.J., Mekler V., Minakhin L., Severinov K. Ebright R.H., and Hochschild A. 2005. The interaction between σ70 and the β-flap of E. coli RNA polymerase inhibits extension of nascent RNA during early elongation. Proc. Natl. Acad. Sci. USA 102: 4488-4493.
Nickels BE, Roberts CW, Roberts JW, and Hochschild A. 2006. RNA-mediated destabilization of the σ70 region 4/β flap interaction facilitates engagement of RNA polymerase holoenzyme by the Q antiterminator. Mol. Cell 24: 457-468.
Deighan P and Hochschild A. 2007. The bacteriophage λ Q antiterminator protein regulates lates gene expression as a stable component of the transcription elongation complex. Mol. Microbiol. 63: 911-920.
Leibman M and Hochschild A. 2007. A σ-core interaction of the RNA polymerase holoenzyme that enhances promoter escape. EMBO J., in press.