The Central Dogma of biologywhereby DMA is replicated, transcribed, and ultimately translated into proteinis carried out by a handful of important enzymes. These enzymes, which include polymerases, helicases, nucleases, and ribosomes, constitute a core group of sophisticated, biomolecular machines. A detailed understanding of such machines is key to any understanding of life itself, and by extension, to the treatment of human disease. Furthermore, progress towards revealing the molecular mechanisms that power Nature's own molecular-scale machines is directly informing much of our current work at the forefront of nanotechnology, which promises to harness the power of nanoscale devices for the betterment of the human condition. Put simply, we need to know how these machines work if we're to fix themor to copy them. A property shared by many nucleic acid enzymes is that they function as processive motors: once bound to their DNA template, they carry out repeated enzymatic cycles, often moving long distances before detaching. This motion is accompanied by the production of force, and requires a continuous input of chemical energy, usually in the form of nucleoside triphosphates. In contrast to classical mechanoenzymes like myosin (which moves muscles) or kinesin (which transports organelles in cells), the motor-like properties of nucleic acid enzymes are continually modulated by the changing information found in the DNA template, yielding a much richer dynamic behavior. Although high-resolution structural data have become available for many important nucleic acid enzymes, comparatively little is understood about the underlying molecular mechanisms. Recently, work on molecular motors has been revolutionized by the ability to measure force and displace- ment at the level of single molecules, using a new generation of biophysical instrumentation, including laser- based optical traps, scanning force microscopy, and advanced fluorescence techniques that can score single photons. Single-molecule studies hold great promise because they supply unique informationparticularly about the distribution and heterogeneity of enzymatic propertiesthat's been largely inaccessible using traditional biochemical or genetic approaches. An assay developed by my group has allowed us to study gene transcription by E. coli RNA polymerase (RNAP) in real time at the level of individual molecules using optical traps. Our prior work with this system has raised specific questions about the elongation mechanism, the load-dependence, the DNA sequence specificity, pausing &stalling behavior, enzyme microstates, enzyme regulation, repair mechanisms, etc.,that we're now in an excellent position to address through continuing study. Single-molecule techniques may even become sensitive enough to measure the size of the individual steps taken by RNAP, which are expected to correspond directly to the spacing of individual bases along the DNA (3.4A). A direct demonstration that RNAP advances in single basepair increments would allow us to rule out a competing theory that it moves by a so-called "inchworm" mechanism.

R01GM057035

1997-09-30

2009-11-30