Dynamics of transcription elongation are finely-tuned by dozens of regulatory factors

Understanding the complex network and dynamics that regulate transcription elongation requires quantitative analysis of RNA polymerase II (Pol II) behavior in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 42 strains of S. cerevisiae, each missing a known elongation regulator including RNA processing factors, transcription elongation factors, histone variants, chromatin modifiers, and chromatin remodelers and chaperones. For each strain, we determined differentially expressed genes, measured levels of antisense transcription, examined sites of high polymerase density, and identified single-nucleotide loci of Pol II pausing. For each elongation phenotype, we found that wild-type Pol II activity sits in the middle of the dynamic range, indicating that elongation is balanced by the opposing effects of many factors. We find that Pol II elongation at sites of RNA processing is co-transcriptionally controlled by key regulators. Furthermore, Pol II pauses frequently across gene bodies at locations that vary across deletion strains. Meta-analysis of all datasets finds that commonly differentially-expressed genes across the deletion strains exhibit more extreme antisense transcription and polymerase pausing, revealing a link between elongation activity and gene regulation. Thus, regulation of transcription elongation impacts co-transcriptional processing and overall gene expression through precisely balancing Pol II activity by a diverse array of factors. Overall design: We deleted 42 known transcription elongation regulators including RNA processing factors, transcription elongation factors, histone variants, chromatin modifiers, chromatin remodelers and chaperones; transcriptional effects of each deletion were assayed using NET-seq. Nearly all mutant samples were analyzed in at least biological duplicate.