The Central Dogma of biology-whereby nucleic acids are replicated, transcribed, and translated into protein-is carried out by a core group of essential enzymes. These enzymes, which include polymerases, helicases, endo/exonucleases, and ribosomes, constitute a set of complex molecular machines. A detailed understanding of such machines is key to understanding life itself, and by extension to the treatment of disease. One property shared by these enzymes is that they function as processive motors: once bound to a DNA or RNA template, they carry out multiple enzymatic cycles. This motion is accompanied by the production of force and requires chemical energy, usually, but not always, in the form of nucleoside triphosphates (NTPs). In contrast to mechanoenzymes such as kinesin, the motor properties of nucleic acid enzymes are continually modulated by information in the template, yielding a rich dynamic behavior. Although high resolution structural data have become available for several nucleic acid enzymes, comparatively little is known about their mechanical properties and mechanisms. Recent work on biological motors has been revolutionized by the ability to monitor force and displacement at the single-molecule level, using biophysical instrumentation combined with in vitro motility assays. Beyond providing new ways of measurement, single molecule studies supply additional kinds of information- particularly about the distribution and heterogeneity of enzyme properties-that are not available with traditional biochemical methods, which yield ensemble averages. An assay for E. coli RNA polymerase now makes it possible to study transcription at the level of individual molecules, and prior work with this system has raised questions about elongation mechanisms, load- dependence, template specificity, pausing/stalling behavior, microstates, etc., that we plan to address through continuing study. We have also successfully developed a single-molecule assay for phage lambda exonuclease (a processive 5' yields 3' directed enzyme that degrades one strand of DNA), which we plan to exploit to learn more about its molecular mechanics, placing special emphasis on the load- and substrate-dependence. Unlike polymerases, or motors such as myosin and kinesin, lambda exonuclease is not powered by NTP hydrolysis, but by energy stored within the DNA itself. It will therefore be of long-term interest to compare and contrast newfound insights into the molecular mechanism of the exonuclease motor with what we learn about RNA polymerase through similar, single-molecule approaches.
| Meng, Cong A; Fazal, Furqan M; Block, Steven M (2017) Real-time observation of polymerase-promoter contact remodeling during transcription initiation. Nat Commun 8:1178 |
| Fazal, Furqan M; Meng, Cong A; Murakami, Kenji et al. (2015) Real-time observation of the initiation of RNA polymerase II transcription. Nature 525:274-7 |
| Schweikhard, Volker; Meng, Cong; Murakami, Kenji et al. (2014) Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms. Proc Natl Acad Sci U S A 111:6642-7 |
| Savinov, Andrew; Perez, Christian F; Block, Steven M (2014) Single-molecule studies of riboswitch folding. Biochim Biophys Acta 1839:1030-1045 |
| Blakely, Brandon L; Dumelin, Christoph E; Trappmann, Britta et al. (2014) A DNA-based molecular probe for optically reporting cellular traction forces. Nat Methods 11:1229-32 |
| Zhou, Jing; Schweikhard, Volker; Block, Steven M (2013) Single-molecule studies of RNAPII elongation. Biochim Biophys Acta 1829:29-38 |
| Koslover, Daniel J; Fazal, Furqan M; Mooney, Rachel A et al. (2012) Binding and translocation of termination factor rho studied at the single-molecule level. J Mol Biol 423:664-76 |
| Palangat, Murali; Larson, Matthew H; Hu, Xiaopeng et al. (2012) Efficient reconstitution of transcription elongation complexes for single-molecule studies of eukaryotic RNA polymerase II. Transcription 3:146-53 |
| Anthony, Peter C; Sim, Adelene Y L; Chu, Vincent B et al. (2012) Electrostatics of nucleic acid folding under conformational constraint. J Am Chem Soc 134:4607-14 |
| Gutiérrez-Medina, Braulio; Andreasson, Johan O L; Greenleaf, William J et al. (2010) An optical apparatus for rotation and trapping. Methods Enzymol 475:377-404 |
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