Single Molecule Studies of Nucleic Acid Enzymes

This chapter reviews the various single molecule methods used and the type of information obtained by such studies of nucleic acid enzymes. Numerous different enzymes have evolved to catalyze the synthesis, digestion, unwinding, and unlinking of nucleic acids that are central to genomic maintenance. The development of single molecule techniques has allowed researchers to study the activity of nucleic acid enzymes, such as RNA polymerases, DNA polymerases, topoisomerases, exonucleases, andDNA helicases, at an unprecedented level of detail. The ability to observe the activity of RNA and DNA-binding proteins on the single molecule level provides tremendous opportunities in the field of nucleic acid enzymology. The chapter provides an overview of the various techniques and a number of nucleic acid enzyme systems where single molecule approaches have proven to be particularly powerful in unraveling the molecular details of enzymatic activity. The recent development of several methods, such as flow stretching, magnetic tweezers, and optical trapping to mechanically manipulate objects of microscopic scale has allowed researchers to stretch and twist individual DNA molecules. Advances in fluorescence spectroscopy and microscopy have made it possible to detect the fluorescence from a single fluorophore under biological tweezers, transcriptional elongation traces of individual E. coliRNA polymerases could be obtained with high spatial resolution, allowing for a detailed study of the statistics of transcriptional pausing. The technical improvements of optical trapping techniques allowed observation of movements of individual RNA polymerases along their template DNA with a resolution better than a single base pair.