Visualizing Transcription In Vivo at Nucleotide Resolution using Nascent Transcript Sequencing

Abstract

Elegant single molecule approaches have elucidated the mechanisms that cause RNA polymerase to pause during transcription. However, it is unclear whether these pausing events and their subsequent recovery occur within the cell as there are a large number of elongation factors that facilitate the progression of RNA polymerase (RNAP) through a chromatized genome. To explore the mechanisms of RNA polymerase elongation in vivo, we require experimental strategies that can observe transcription with the same resolution as can be obtained in vitro. Here we present an approach, native elongating transcript sequencing - NET-seq, that accomplishes this goal by exploiting the extraordinary stability of the DNA-RNA-RNAP ternary complex to capture nascent transcripts directly from live cells without crosslinking. The identity and abundance of the 3’ end of purified transcripts are revealed by deep sequencing thus providing a quantitative measure of RNAP density with single nucleotide precision. Application of NET-seq in Saccharomyces cerevisiae reveals pervasive polymerase pausing and backtracking throughout the body of transcripts. Average pause density shows prominent peaks at each of the first four nucleosomes with the peak location occurring in good agreement with in vitro single molecule measurements. Thus nucleosome-induced pausing represents a major barrier to transcriptional elongation in vivo.