Separate domains of fission yeast Cdk9 (P-TEFb) are required for capping enzyme recruitment and primed (Ser7-phosphorylated) Rpb1 carboxyl-terminal domain substrate recognition

Mol Cell Biol. 2012 Jul;32(13):2372-83. doi: 10.1128/MCB.06657-11. Epub 2012 Apr 16.

Abstract

In fission yeast, discrete steps in mRNA maturation and synthesis depend on a complex containing the 5'-cap methyltransferase Pcm1 and Cdk9, which phosphorylates the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) and the processivity factor Spt5 to promote transcript elongation. Here we show that a Cdk9 carboxyl-terminal extension, distinct from the catalytic domain, mediates binding to both Pcm1 and the Pol II CTD. Removal of this segment diminishes Cdk9/Pcm1 chromatin recruitment and Spt5 phosphorylation in vivo and leads to slow growth and hypersensitivity to cold temperature, nutrient limitation, and the IMP dehydrogenase inhibitor mycophenolic acid (MPA). These phenotypes, and the Spt5 phosphorylation defect, are suppressed by Pcm1 overproduction, suggesting that normal transcript elongation and gene expression depend on physical linkage between Cdk9 and Pcm1. The extension is dispensable, however, for recognition of CTD substrates "primed" by Mcs6 (Cdk7). On defined peptide substrates in vitro, Cdk9 prefers CTD repeats phosphorylated at Ser7 over unmodified repeats. In vivo, Ser7 phosphorylation depends on Mcs6 activity, suggesting a conserved mechanism, independent of chromatin recruitment, to order transcriptional CDK functions. Therefore, fission yeast Cdk9 comprises a catalytic domain sufficient for primed substrate recognition and a multivalent recruitment module that couples transcription with capping.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain
  • Cyclin-Dependent Kinase 9 / chemistry*
  • Cyclin-Dependent Kinase 9 / genetics
  • Cyclin-Dependent Kinase 9 / metabolism*
  • Enzyme Activation
  • Genes, Fungal
  • Methyltransferases / chemistry
  • Methyltransferases / genetics
  • Methyltransferases / metabolism
  • Models, Biological
  • Mutation
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / metabolism*
  • Phosphorylation
  • Positive Transcriptional Elongation Factor B / chemistry*
  • Positive Transcriptional Elongation Factor B / genetics
  • Positive Transcriptional Elongation Factor B / metabolism*
  • Protein Interaction Domains and Motifs
  • RNA Polymerase II / chemistry*
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / growth & development
  • Schizosaccharomyces / metabolism*
  • Schizosaccharomyces pombe Proteins / chemistry*
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism*
  • Serine / chemistry
  • Substrate Specificity
  • Transcriptional Elongation Factors / chemistry
  • Transcriptional Elongation Factors / genetics
  • Transcriptional Elongation Factors / metabolism

Substances

  • Schizosaccharomyces pombe Proteins
  • Spt5 protein, S pombe
  • Transcriptional Elongation Factors
  • Serine
  • Methyltransferases
  • mRNA (guanine(N7))-methyltransferase
  • Positive Transcriptional Elongation Factor B
  • Cyclin-Dependent Kinase 9
  • Nucleotidyltransferases
  • RNA Polymerase II
  • mRNA guanylyltransferase