In eukaryotes, the transcription cycle, which includes initiation, elongation, and termination, is regulated at multiple steps by both cis-regulatory elements and trans-acting factors. At protein-coding genes, successful transcription initiation is often followed by a pivotal regulatory step, i.e. the promoter-proximal pause. During this pause, the elongation complex is supplemented by additional elongation factors enabling productive elongation, proper termination and co-transcriptional RNA processing. In addition to this scheduled regulated pause, Pol II encounters obstacles such as nucleosomes, pause predisposing cis- sequences or other physical barriers that cause Pol II to move backward - a phenomenon referred to as Pol II backtracking. In case of a transient pause, when Pol II backtracks by one nucleotide, it can cleave the backtracked nucleotide itself, as it has an intrinsic nuclease property. However, when more extensive backtracking is thermodynamically favorable, Pol II is trapped in an immobilized state known as transcriptional arrest and requires an auxiliary factor TFIIS (TCEA in human cells). TFIIS alleviates transcriptional arrest by enhancing the RNA cleavage activity of Pol II. Following the hydrolysis of backtracked RNA, a new 3’ is generated and the active site of Pol II is free to resume RNA synthesis. The molecular mechanism of TFIIS has been extensively characterized both structurally and biochemically; however, our understanding of its role in human cells remains incomplete because of the additional complexity incurred by four highly conserved TFIIS paralogs. To date, there have been no multiomics studies investigating paralog-specific roles. In our research project, we analyzed the function of two paralogs expressed in HEK293T cells: the ubiquitously expressed TCEA1 and lowly expressed cerebellum- and testis- specific TCEA2. We employed two cellular systems: an inducible epitope-tagged protein expression system to determine their individual interaction partners and genomic binding patterns and CRISPR- knockouts of each and both proteins to evaluate the impact on elongation at the level of nascent and messenger RNA. Our results demonstrate that both paralogs bind Pol II shortly after initiation and, in their absence, Pol II occupancy is strikingly increased at the promoter-proximal region of the majority of active genes, as revealed by High-sensitive native elongating transcript sequencing (HiS-NET-seq). About a half of those genes have reduced Pol II occupancy in the gene body. This indicates that backtracking is a common event at the promoter-proximal region and TCEA1 and TCEA2 are needed to progress into the gene body and, possibly, also for proper Pol II processivity. Unexpectedly, TCEA2 deletion was more detrimental to cellular growth than the TCEA1 knockout. The TCEA2 and the double knockout cells had a profound delay in DNA replication accompanied by activated DNA damage response. The deletion of TCEA2 had a more pronounced impact on the transcriptional output, indicating that, despite its higher expression level, TCEA1 alone is not sufficient to alleviate backtracking. This suggests that TCEA2 is a potent elongation factor and has its own target genes that regulate cell proliferation.
