How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes

Genes Dev. 2014 Jun 15;28(12):1323-36. doi: 10.1101/gad.242768.114.

Abstract

Interactions between RNA guanylyltransferase (GTase) and the C-terminal domain (CTD) repeats of RNA polymerase II (Pol2) and elongation factor Spt5 are thought to orchestrate cotranscriptional capping of nascent mRNAs. The crystal structure of a fission yeast GTase•Pol2 CTD complex reveals a unique docking site on the nucleotidyl transferase domain for an 8-amino-acid Pol2 CTD segment, S5PPSYSPTS5P, bracketed by two Ser5-PO4 marks. Analysis of GTase mutations that disrupt the Pol2 CTD interface shows that at least one of the two Ser5-PO4-binding sites is required for cell viability and that each site is important for cell growth at 37°C. Fission yeast GTase binds the Spt5 CTD at a separate docking site in the OB-fold domain that captures the Trp4 residue of the Spt5 nonapeptide repeat T(1)PAW(4)NSGSK. A disruptive mutation in the Spt5 CTD-binding site of GTase is synthetically lethal with mutations in the Pol2 CTD-binding site, signifying that the Spt5 and Pol2 CTDs cooperate to recruit capping enzyme in vivo. CTD phosphorylation has opposite effects on the interaction of GTase with Pol2 (Ser5-PO4 is required for binding) versus Spt5 (Thr1-PO4 inhibits binding). We propose that the state of Thr1 phosphorylation comprises a binary "Spt5 CTD code" that is read by capping enzyme independent of and parallel to its response to the state of the Pol2 CTD.

Keywords: RNA polymerase II; Spt5; mRNA capping; regulation; transcription.

Publication types

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

MeSH terms

  • Genetic Code
  • Models, Molecular*
  • Nucleotidyltransferases / chemistry
  • Nucleotidyltransferases / metabolism*
  • Phosphorylation
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • RNA Polymerase II* / chemistry
  • RNA Polymerase II* / genetics
  • RNA Polymerase II* / metabolism
  • Schizosaccharomyces / enzymology*
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / metabolism
  • Schizosaccharomyces pombe Proteins* / chemistry
  • Schizosaccharomyces pombe Proteins* / genetics
  • Schizosaccharomyces pombe Proteins* / metabolism
  • Threonine / metabolism
  • Transcriptional Elongation Factors* / chemistry
  • Transcriptional Elongation Factors* / genetics
  • Transcriptional Elongation Factors* / metabolism

Substances

  • Schizosaccharomyces pombe Proteins
  • Spt5 protein, S pombe
  • Transcriptional Elongation Factors
  • Threonine
  • Nucleotidyltransferases
  • RNA Polymerase II
  • mRNA guanylyltransferase