PHYS 114 |
| T7 RNA polymerase (RNAP) represents a simple (single subunit) system to understand the complex process via which RNAP achieves transcription initiation. T7 RNAP-DNA complex undergoes several changes as it transforms from initiation complex (IC) undergoing abortive synthesis (synthesis of 8-12 RNA transcripts) to a processive enzyme (elongation complex (EC) ) capable of generating RNA thousands of bases in length. Crystal structures of the T7 RNAP-DNA complex with the transcript RNA as initiation complex and elongation complex have already revealed that this transition is made possible by large conformation changes in the protein, release of the promoter region of the DNA and creation of a RNA exit pore. Several biochemical studies have been filling in the gaps between the two ?crystallographic? snapshots trying to determine the exact nature of this transition. We have employed single molecule FRET to investigate transcription initiation by single T7 RNA polymerases to this end. By stalling transcription at a particular position on the DNA template, one can measure FRET on DNA labeled with a FRET pair. Our results show that DNA template is increasingly being packed into the RNAP while it synthesizes RNA upto +8 position (with respect to transcription start site). Beyond +8 position, we observe recovery of the DNA contour extension that is consistent with promoter release. We also examined conformational changes in the protein as we ?walk? the RNA polymerase along the DNA template using donor labeled RNAP. We observe a drastic change in the subdomain H in the N-terminal as the protein rearranges itself when transcription proceeds beyond position +8. These results are consistent with the crystal structures and other bulk techniques and give us a unique experimental handle to look at the dynamics of transcription initiation. |
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Frontiers in Single-Molecule Biophysical Chemistry and Imaging
8:20 AM-12:00 PM, Monday, 11 September 2006 Grand Hyatt San Francisco -- Plaza Ballroom East, Oral
Division of Physical Chemistry |