Mathematical Biology Seminar

L. Stirling Churchman
UC San Francisco
3:05PM, Wednesday, November 17, 2010
LCB 225
Visualizing transcription of RNA at single nucleotide resolution in vivo

Abstract: Despite advances in rapid genome sequencing, fundamental questions remain on how information encoded in DNA is accessed by the cell. It is well known that DNA is transcribed nucleotide by nucleotide into RNA by the molecular machine, RNA polymerase. However, in recent years the assumption that only genes are transcribed has been challenged with the perplexing discovery that many, if not most, RNA transcripts are rapidly degraded and are not from genes. These observations motivate creating approaches that allow a high resolution view of RNA polymerase activity across the genome. Furthermore, such techniques would provide insight into how transcription occurs through nucleosomes - the organizing units of DNA. A longstanding paradox in cell biology is that the enormity of the genome requires it to be organized into a compact 3D structure, yet it must remain accessible to transcriptional activity. We have developed an approach, native elongating transcript sequencing (NET-seq), that uses deep sequencing technology to identify where all nascent RNA transcripts are being made with nucleotide resolution. The ability to define precisely where transcription occurs allowed us to investigate how spurious transcripts are created. Moreover, as NET-seq measurements are quantitative, these data reveal the density of RNA polymerase at nucleotide resolution indicating locations where a pause is likely to occur due to a transcriptional barriers. Our data show pervasive RNA polymerase pausing throughout the bodies of genes. Pause density peaks at nucleosomes, with the peak location occurring in good agreement with biophysical measurements made in single molecule optical trapping experiments. Thus nucleosomes represent a major barrier to transcription. We show here that NET-seq experiments can reveal how regions of the genome are chosen to be transcribed and the physical basis of transcription through a highly organized genome.
For a description of research click here