Spt5's central KOW domains and the Pol II stalk collaborate to regulate chromatin and 3'-end processing in...

Spt5 is a universally conserved multidomain transcription elongation factor that acts as a component of all Pol II elongation complexes (ECs). Structural studies indicate that several of Spt5's central KOW domains lie adjacent to the Pol II stalk, composed of subunits Rpb4 and Rpb7. However, the in vivo functions of Spt5's central KOW domains are unknown. Here, we show that Spt5 and Rpb4/7 jointly modulate 3'-end formation and co-transcriptional chromatin integrity in Saccharomyces cerevisiae. We identify mutations in the SPT5 KOW2-3 domains and RPB7 that cause cryptic initiation of transcription and alter 3'-end formation of RNA transcripts. Molecular readthrough assays reveal allele-specific changes at both GAL10 and SNR13, consistent with impacts on termination mediated by Cleavage and Polyadenylation Factor (CPF)-Cleavage Factor (CF) and the Nrd1-Nab3-Sen1 (NNS) pathway. Proteomic experiments with isolated KOW domains enrich factors from both pathways as well as chromatin regulators, overlapping known Rpb7 interactors. Together, these findings support a model in which the Spt5 KOW2-4/Pol II stalk region acts as a recruitment platform that coordinates pre-mRNA processing and chromatin dynamics during elongation, revealing new roles for the central KOW domains of Spt5.