The Central Dogma of biology-whereby genetic information in DNA is replicated, transcribed, and ultimately translated into protein-is carried out by a handful of essential biomolecules that include polymerases, helicases, isomerases, and ribosomes. Together, these enzymes constitute a core group of sophisticated, bio- molecular machines. Understanding their function holds a key to understanding life, and by extension, to the treatment of human disease. Hard-won knowledge about biomolecules is informing work at the forefront of nanoscience, which hopes to harness tiny manmade devices to better the human condition. Put simply, we need to know how Nature's own machines work if we9re ever to fix them or to emulate them. A property shared by many nucleic acid-based enzymes is that they function as motors: once bound to DNA or RNA, they undergo repeated enzymatic cycles, often traveling considerable distances. This processive motion is ac- companied by force production and requires chemical energy. In contrast to classic mechanoenzymes like myosin, the properties of nucleic acid-based motors are modulated by an ever-changing template underfoot, yielding rich behavior. Although structural data are available for many such enzymes, comparatively little is known about their fundamental mechanisms. Recently, biophysical studies have been revolutionized by the ability to measure force and displacement at the level of individual molecules, using a variety of new techniques that include optical traps, nanometry, and fluorescence. Single-molecule techniques can supply critical information hitherto inaccessible to traditional approaches. In the previous grant cycle, my group succeeded in developing high-resolution optical trapping instrumentation that is able to register displacements down to the atomic level (~1E). Consequently, we can record from single bacterial RNA polymerase (RNAP) molecules as these step from base to base along DNA. Base-pair resolution makes it possible to sequence DNA based on enzyme motion, and points to new directions in nanoscience. Improved instrumental stability also allows us to reconstruct energy landscapes for folding transitions in nucleic acids that form complex structures (hairpins, ribozymes, etc.). We propose to continue with our single-molecule work on transcription by RNAP. Addition- ally, we plan to use a variant of the single-molecule assay for RNAP to address unsolved problems of co- transcriptional folding and gene regulation, particularly by riboswitches formed in nascent mRNAs, and to better understand the sequence elements that regulate transcriptional elongation and termination. A related assay will allow us to study the initiation of translation by ribosomes in eukaryotes, together with the sequence elements that modulate that process. Finally, we developed a successful single-molecule assay for transcriptional elongation by Pol II, the eukaryotic homolog of bacterial RNAP. We plan to capitalize on this opportunity by studying Pol II molecules purified from calf thymus and yeast, and to compare and contrast their biophysical properties with one another, as well as with those of prokaryotic RNAP.
Understanding the process of gene regulation is fundamental to any understanding of disease, because undesired changes in gene expression are responsible for the overwhelming majority of developmental and inherited disorders. Furthermore, many communicable diseases, especially those caused by human viruses, typically involve disruptions of gene regulation produced by the pathogen itself. The work described in this research proposal will supply new insights into the molecular basis of gene regulation by studying, at the single-molecule level, important gene- control molecules such as RNA polymerases, ribosomes, ribozymes, and riboswitches.
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