Phosphorylation of the Pol II CTD by KIN28 Enhances BUR1/BUR2 Recruitment and Ser2 CTD Phosphorylation Near Promoters

Abstract

Cyclin-dependent kinase BUR1/BUR2 appears to be the yeast ortholog of P-TEFb, which phosphorylates Ser2 of the RNA Pol II CTD, but the importance of BUR1/BUR2 in CTD phosphorylation is unclear. We show that BUR1/BUR2 is co-transcriptionally recruited to the 5′ end of ARG1 in a manner stimulated by interaction of the BUR1 C-terminus with CTD repeats phosphorylated on Ser5 by KIN28. Impairing BUR1/BUR2 function, or removing the CTD-interaction domain in BUR1, reduces Ser2 phosphorylation in bulk Pol II and eliminates the residual Ser2P in cells lacking the major Ser2 CTD kinase, CTK1. Impairing BUR1/BUR2 or CTK1 evokes a similar reduction of Ser2P in Pol II phosphorylated on Ser5, and in elongating Pol II near the ARG1 promoter. By contrast, CTK1 is responsible for the bulk of Ser2P in total Pol II and at promoter-distal sites. In addition to phosphorylating Ser2 near promoters, BUR1/BUR2 also stimulates Ser2P formation by CTK1 during transcription elongation.

Two Distinct Roles of ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) at Promoters and within Transcribed Regions of ATX1-Regulated Genes

“…The rapid addition of Ser5P often results in the detection of this modification in promoter regions (Gomes et al, 2006;Chapman et al, 2007;Kim et al, 2009;Qiu et al, 2009), but the detection of Ser2P at the promoter region of the genes was unusual. To examine whether the signals might originate from lack of specificity of the antibodies, we investigated the specificities of both the Ser5P and Ser2P antibodies against synthetic peptides.…”

“…Ser2P is mediated by CDK9, which is recruited via a Ser5P-dependent mechanism (Qiu et al, 2009). The Ser2P modification recruits factors for mRNA polyadenylation and termination (McCracken et al, 1997;Birse et al, 1998).…”

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

“…These findings are reminiscent to observations in fission yeast where initial phosphorylation of the CTD by the kinase Mcs6 of TFIIH stimulates subsequent phosphorylation by P-TEFb, possibly to couple elongation and capping of select pre-mRNAs 55 . Likewise, the cyclin-dependent kinase Bur1/Bur2, which is the budding yeast orthologue of human Cdk9, was shown to directly stimulate Ser5 and indirectly Ser2 phosphorylation by Ctk1 during transcriptional elongation 56 . Other CTD-modifying enzymes, such as the two recently identified Cdk12 and Cdk13 kinases 41,42 , may additionally contribute to the variety and spatial or temporal preferences of CTD phosphorylations.…”

A role for phosphorylated Pol II CTD in modulating transcription coupled histone dynamics

“…Together, these results indicated that while H3K36 me2/3 was required for Rpd3C(S) binding to nucleosomes, some other factor was responsible for its recruitment to chromatin. Because various factors involved in elongation or mRNA processing, including Set2, were shown to be recruited to chromatin through elongating Pol II, 4,5,[20][21][22][23] we sought to determine whether Rpd3C(S) physically interacted with Pol II, phosphorylated at the C-terminal Domain (CTD). Co-IP experiments revealed that Rco1 co-immunoprecipitated with Ser5 phosphorylated (Ser5P) likely transferred by RSC or SWI/SNF to histone chaperones such as Asf1 or FACT which can then aid in reassembly.…”