Abstract
A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)=2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for)=0.36 s(-1)), and the processive synthesis (k(pol)=43 nt s(-1)) and release of a gene-length RNA transcript (similar to1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off)>k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape.
| Original language | English |
|---|---|
| Pages (from-to) | 3239-3244 |
| Number of pages | 6 |
| Journal | Journal of Biological Chemistry |
| Volume | 279 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - 30 Jan 2004 |
Keywords
- OPEN COMPLEX-FORMATION
- T7-RNA POLYMERASE
- REAL-TIME
- KINETIC MECHANISM
- DNA
- PURIFICATION
- FORCE
- PROCESSIVITY
- FLUORESCENCE
- SPECIFICITY