The fidelity of transcription in human cells.

To determine the error rate of transcription in human cells, we analyzed the transcriptome of H1 human embryonic stem cells with a circle-sequencing approach that allows for high-fidelity sequencing of the transcriptome. These experiments identified approximately 100,000 errors distributed over every major RNA species in human cells. Our results indicate that different RNA species display different error rates, suggesting that human cells prioritize the fidelity of some RNAs over others.

A Transcription Fidelity Reporter Identifies GreA as a Major RNA Proofreading Factor in .

We made a coupled genetic reporter that detects rare transcription misincorporation errors to measure RNA polymerase transcription fidelity in Using this reporter, we demonstrated that the transcript cleavage factor GreA, but not GreB, is essential for proofreading of a transcription error where a riboA has been misincorporated instead of a riboG. A mutant strain had more than a 100-fold increase in transcription errors relative to wild-type or a mutant. However, overexpression of GreB in Δ cells reduced the misincorporation errors to wild-type levels, demonstrating that GreB at high concentration could substitute for GreA in RNA proofreading activity .

RNA polymerase II senses obstruction in the DNA minor groove via a conserved sensor motif.

RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation.