Identification of novel functions of the Paf1C and Npl3 during RNA polymerase II transcription elongation

The process by which information stored within DNA is transmitted to the cellular machinery is through the synthesis of RNA transcripts, which is performed by DNA-dependent RNA polymerases. Transcription by RNA polymerase II (RNAPII) is composed of three main stages: initiation, elongation, and termination. Accessory factors regulating elongation perform a variety of functions, including facilitating RNAPII's passage through chromatin and maturation of the RNA. In this dissertation, we further characterize the molecular mechanisms regulating two factors that function during RNAPII elongation. The Paf1 complex (Paf1C) is involved in a variety of processes during elongation, however it is unknown if the Paf1C can directly affect the elongation activity of RNAPII. In Chapter Two, we demonstrate that the Paf1C from Saccharomyces cerevisiae does not stimulate elongation by RNAPII in vitro . Interestingly, in vivo the Paf1C localizes primarily to the open reading frames of genes, suggesting that the presence of the RNA transcript promotes its localization. We discover that the Paf1C binds RNA, and this interaction stabilizes the complex's localization at transcribed genes. Additionally, we identify Leo1 and Rtf1, two of the Paf1C subunits, as posessing RNA binding activity, however Leo1 significantly contributes to the complex's association with RNA. Additionally, yeast strains lacking Leo1 display decreased occupancy of histone H3 within actively transcribed genes, indicating that Leo1 is important for Paf1C localization and participates in maintaining proper chromatin structure during transcription. The RNA export factor Npl3 also associates with the RNA transcript during elongation. In Chapter Three we examine Npl3's ability to affect the elongation activity of RNAPII to further investigate Npl3's function as an anti-terminator. Our data indicate that Npl3 physically interacts with RNAPII and stimulates in vitro elongation by RNAPII, and both these activities are inhibited by phosphorylation of Npl3. We demonstrate that the yeast kinase Cka1 phosphorylates Npl3, resulting in reducing Npl3's ability to effectively compete with the RNA processing factor Rna15 for binding to RNA. Additionally, we determined that mutation of the phosphorylated residue results in termination defects in vivo , indicating that phosphorylation of Npl3 is necessary for efficient termination.