PUBLIC INFORMATION FOR:

J. Shaevitz, E. Abbonanzieri, R. Landick, S. Block Nature 426: 684 - 687 (2003) (Full Text PDF).

Backtracking by single RNA polymerase molecules observed at near-base-pair resolution.

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Summary

RNA polymerase, the enzyme that copies DNA into RNA in a process termed transcription, achieves remarkable fidelity by actively editing the message it is transcribing. When it makes a mistake (i.e., it inserts the wrong base -- A,C,G or U -- into the RNA chain), the polymerase is able to back up along the DNA and cut off the end portion of the newly-synthesized RNA containing the incorrectly-incorporated base. It then can re-start RNA synthesis in the normal direction, adding bases onto the new end of the RNA chain. By using an ultra-stable optical trapping apparatus, we report the first direct observation of backtacking by single RNA polymerase molecules. On average, we find that once every ~1,000 bases, RNA polymerase stops moving forwards and backs up by about five bases, typically pausing for greater than twenty seconds. (For comparison, the distance corresponding to a single base pair of DNA is just 3.4 angstroms, or about one hundred-millionth of an inch. RNA polymerase normally transcribes DNA at a rate of ~15 bases per seconds, which is about the rate that human hair grows).

By adding the nucleotide analogue inosine, which is similiar to guanosine but which forms a weaker Watson-Crick pair with cytosine, we were able to induce transcriptional errors, raising the frequency of the backtracking pauses by a factor of two. To efficiently remove the incorrectly-incorporated nucleotide from the RNA, the polymerase requires the action of the transcription factors GreA and GreB. These proteins stimulate the cleavage of the newest RNA bases once the polymerase is backtracked. We find that the addition of these proteins greatly reduces the duration of the backtracked pauses, allowing the polymerase to continue transcription, thereby correcting the mistake.

 

Images and Movies

ALL IMAGES ARE COPYRIGHTED AND MAY BE USED WITH PERMISSION.
CONTACT: Prof.. Steven Block sblock@stanford.edu

1. The RNA polymerase proofreading mechanism (Credit: E.. Abbondanzieri)

[Proofreading mechanism.jpg 261 KB]

2. Dumbell optical trapping setup for studying RNA polymerase (Credit: E. Abbondanzieri)

[Dumbell Optical Trapping Cartoon.jpg 263 KB]

3. Photo of the optical trapping instrument used to measure RNA polymerase backtracking (Credit J. Shaevitz)

[Optical Trap Photo.jpg 525 KB]

4. Movie of RNA polymerase motion showing no long pauses, sped up 30 times. (Credit: J. Shaevitz and E. Abbondanzieri)

[RNA polymerase no pause movie.avi 178 KB]

5. Movie of RNA polymerase motion demonstrating a five minute pause, sped up 60 times. (Credit: J. Shaevitz and E. Abbondanzieri)

[RNA polymerase long pause movie.avi 783 KB]

6. Record of RNA polymerase motion taken during above movie (no long pause). (Credit: J. Shaevitz and E. Abbondanzieri)

[RNA polymerase no pause record.jpg 182 KB]

7. Record of RNA polymerase motion taken during above movie (long pause). (Credit: J. Shaevitz and E. Abbondanzieri)

[RNA polymerase long pause record.jpg 182 KB]

 


Steven M. Block
Prof. of Applied Physics & Biological Sciences
Stanford University
Phone: 650-724-4046
Email: sblock@stanford.edu

 

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