Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

Abstract

Schizosaccharomyces pombe Cdk9/Pch1 protein kinase is a functional ortholog of the essential Saccharomyces cerevisiae Bur1/Bur2 kinase and a putative ortholog of metazoan P-TEFb (Cdk9/cyclin T). SpCdk9/Pch1 phosphorylates of the carboxyl-terminal domain (CTD) of the S. pombe transcription elongation factor Spt5, which consists of 18 tandem repeats of a nonapeptide of consensus sequence 1 TPAWNSGSK 9 . We document the divalent cation dependence and specificity of SpCdk9/Pch1, its NTP dependence and specificity, the dependence of Spt5-CTD phosphorylation on the number of tandem nonamer repeats, and the specificity for phosphorylation of the Spt5-CTD on threonine at position 1 within the nonamer element. SpCdk9/Pch1 also phosphorylates the CTD heptaptide repeat array of the largest subunit of S. pombe RNA polymerase II (consensus sequence YSPTSPS) and does so exclusively on serine. SpCdk9/Pch1 catalyzes autophosphorylation of the kinase and cyclin subunits of the kinase complex. The distribution of phosphorylation sites on SpCdk9 (86% Ser(P), 11% Thr(P), 3% Tyr(P)) is distinct from that on Pch1 (2% Ser(P), 98% Thr(P)). We conducted a structure-guided mutational analysis of SpCdk9, whereby a total of 29 new mutations of 12 conserved residues were tested for in vivo function by complementation of a yeast bur1⌬ mutant. We identified many lethal and conditional mutations of side chains implicated in binding ATP and the divalent cation cofactor, phosphoacceptor substrate recognition, and T-loop dynamics. We surmise that the lethality of the of T212A mutation in the T-loop reflects an essential phosphorylation event, insofar as the conservative T212S change rescued wild-type growth; the phosphomimetic T212E change rescued growth at 30°C; and the effects of mutating the T-loop threonine were phenocopied by mutations in the three conserved arginines predicted to chelate the phosphate on the T-loop threonine.mRNA synthesis by RNA polymerase (pol) 1 II is regulated by phosphorylation of several of the protein components of the transcription apparatus (1). Phosphorylation of the carboxylterminal domain (CTD) of the largest subunit of pol II has been the focus of much attention because the CTD acts as an essential scaffold for the binding of macromolecular assemblies that regulate mRNA synthesis and cotranscriptional mRNA processing (2). The pol II CTD is composed of a heptapeptide motif of consensus sequence YSPTSPS repeated in tandem. The mammalian pol II CTD has 52 heptad repeats, the fission yeast Schizosaccharomyces pombe CTD has 29 repeats, and the budding yeast Saccharomyces cerevisiae CTD has 26 copies. The pol II CTD undergoes a cycle of extensive phosphorylation and dephosphorylation at positions Ser 5 and Ser 2 , which is coordinated with the transcription cycle (3). Multiple CTD kinases are present in eukaryotic cells, e.g. budding yeast has four CTD kinases, two of which (Kin28 and Bur1) are essential (3). CTD kinases are heteromeric enzymes consisting of a cyclin subunit plus a cyclin-dependent kinase (Cdk) subun...

RNA Polymerase II Elongation Control

“…Phosphorylation of one of the four NELF subunits (NELFe) has also been linked with its removal from Pol II during the transition into productive elongation (54). Even though yeast do not have NELF or poised polymerases, the Bur1/2 kinase has been demonstrated to phosphorylate DSIF and seems to be performing a similar function to transition the polymerase into productive elongation (55, 56). …”

RNA Polymerase II transcription elongation and Pol II CTD Ser2 phosphorylation

“…28 In contrast, Bur1 is important for normal growth as both bur1 and bur2 null mutants are extremely slow growing. 31,35 Similarly, in fission yeast, mutants of the Ctk1 homolog, Lsk1, are also viable under normal growth conditions, 36 while mutants of the Bur1 homolog, Cdk9, are lethal 37 ( Fig. 1A).…”

A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS

“…A region of NELF-A that resembles hepatitis Ѩ antigen binds RNAPII (8), whereas a C-terminal region of RD (NELF-E) binds RNA (9) and is proposed to mediate inhibition of transcription through interaction with nascent RNA as the RNA emerges from the RNAPII exit channel (8). Both SPT5 and RD (NELF-E) also can be phosphorylated by P-TEFb (10)(11)(12), which may contribute to the formation of productive TECs.…”