The Central Dogma of Biology-whereby genetic information gets replicated, transcribed, and translated into proteins -- is carried out by a handful of critical enzymes that include polymerases, helicases, and ribosomes. Together, these enzymes constitute a core group of sophisticated, biomolecular 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 devices to better the human condition. Put simply, we need to know how Nature's machines work if we're ever to fix them or to emulate them. A property shared by many nucleic acid-based enzymes is that they function as molecular motors: once bound to DNA or RNA, they undergo repeated cycles, often traveling considerable distances. This motion is accompanied by force production and requires chemical energy. In contrast to 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 molecular mechanisms. Recently, biophysical studies have been revoliitionized by the ability to measure forces and displacements at the level of single molecules, using techniques that include optical traps, nanometry, and fluorescence. Single-molecule approaches supply critical information that has been hitherto inaccessible by traditional techniques. Previously, my group succeeded in developing optical trapping instrumentation that's able to register displacements down to the atomic level (~1 A). Consequently, we can record from bacterial RNA polymerase (RNAP) molecules as these step from base to base along DNA. Improved instrumental stability now allows us to reconstruct energy landscapes for folding transitions in nucleic acids that form complex structures (riboswitches, ribozymes, etc.). We propose to continue our single-molecule work on transcription by RNAP. We also plan to use single-molecule assays to address unsolved problems of co-transcriptional folding and gene regulation, and to better understand the sequence elements that regulate transcriptional elongation and termination (e.g., riboswitches). A closely related assay wiU allow us to study the initiation of translation by ribosomes in eukaryotes, along with the RNA sequence elements that modulate that process. Finally, we will pursue a successful single-molecule assay we developed for transcription by Pol II (RNAPII), the eukaryotic analog of RNAP.
(See Instructions): As stated in the Project Summary, understanding the function of the core set of biomolecules responsible for transmitting genetic information is fundamental to an understanding life itself, and by extension; to the treatment of human disease. We need to know how Nature's molecular machinery actually works if we're ever to fix it when it's broken (a central goal in medicine), or to emulate it in the development of new manmade devices (a central goal in nanoscience). This project seeks some of that understanding.
| Milic, Bojan; Chakraborty, Anirban; Han, Kyuho et al. (2018) KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics. Proc Natl Acad Sci U S A 115:E4613-E4622 |
| Savinov, Andrew; Block, Steven M (2018) Self-cleavage of the glmS ribozyme core is controlled by a fragile folding element. Proc Natl Acad Sci U S A 115:11976-11981 |
| 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; Koslover, Daniel J; Luisi, Ben F et al. (2015) Direct observation of processive exoribonuclease motion using optical tweezers. Proc Natl Acad Sci U S A 112:15101-6 |
| 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 |
| García-García, Cuauhtémoc; Frieda, Kirsten L; Feoktistova, Kateryna et al. (2015) RNA BIOCHEMISTRY. Factor-dependent processivity in human eIF4A DEAD-box helicase. Science 348:1486-8 |
| Duesterberg, Van K; Fischer-Hwang, Irena T; Perez, Christian F et al. (2015) Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer. Elife 4: |
| 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 |
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