CPM Seminar

Visualizing RNA transcription in cells at single nucleotide resolution

Stirling Churchmann

Harvard University

Non-technical abstract:
Cells decode their genomes with a large molecular machine through a process called transcription. However genomic DNA is coated with proteins and it is not clear how transcription reacts to these barriers. We developed an experimental approach that observes transcription across the genome at 3.4 angstrom resolution using massively parallel sequencing technology. Our observations demonstrate that transcription is strongly modulated by protein obstacles.

Technical abstract:
The cell decodes genomic information starting with the process of transcription where the molecular machine, RNA polymerase, creates an RNA molecule using the DNA genome as a template. The RNA molecule is a temporary copy of a portion of the DNA that is processed to remove large regions through a splicing chemical reaction. RNA polymerase has to navigate a genome that is organized into structures called nucleosomes and is bound by many proteins. In order to understand how RNA polymerase progresses through DNA in living cells, we developed an approach, native elongating transcript sequencing (NET-seq),that uses deep sequencing technology to identify the location of RNA polymerase with nucleotide resolution. Our NET-seq data reveal the average density of RNA polymerase across the genome indicating locations where pausing typically occurs due to barriers. Pause density peaks at nucleosomes, with the peak location occurring in good agreement with biophysical measurements made in single molecule optical trapping experiments. Pausing also occurs at sites where DNA-binding proteins are located. We also see high density of RNA polymerase at locations where the RNA is spliced, suggesting that pausing contributes to the processing of RNA. Together, NET-seq data reveal the physical basis of transcription through a highly organized genome.