| Budget Amount *help |
¥14,100,000 (Direct Cost: ¥14,100,000)
Fiscal Year 2000: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1999: ¥10,000,000 (Direct Cost: ¥10,000,000)
|
|---|
| Research Abstract |
RNA polymerase plays a key role in the transcription of gene expression by synthesizing RNA transcripts from DNA templates. To transcribe DNA, RNA polymerase moves along the DNA helix. Whether RNA polymerase-precisely follows DNA helix is an unanswered question bearing directly on the mechanism. To observe relative rotation between RNAP and DNA, we employed optical microscopy based on the tethered-particle method. A DNA template, 4971 base-pair (1.7 μm) long and containing one strong promoter was constructed. The complex of polymerase and DNA was attached to a glass surface and a magnetic bead of diameter 850 nm coated with streptavidin was attached to the downstream end of the DNA where nine nucleotide residues were biotinylated. To observe rotation, we decorated the end bead with smaller fluorescent beads. We pulled the magnetic bead upward at 〜0.1 pN with a magnet for two reasons : to confine the rotation in a horizontal plane, and in the hope of restraining the DNA from supercoiling, which would interfere with torque transmission to the end bead. Then, all four nucleoside triphosphates were added to allow transcription. Up to a few percent of beads in an observation chamber rotated continuously invariably clockwise. Threading a right-handed double helix of DNA through RNAP will, in a simple mechanism, produce clockwise rotation. When NTPs were absent, unidirectional rotation was not observed. Our results indicate that RNAP rotates DNA by tracking its right-handed helix, that RNAP does so over thousands of base pairs, and that RNAP can produce >5 pN nm of torque. The real-time observation of rotation opens the possibility of resolving individual transcription steps.
|
