Fission yeast RNA triphosphatase reads an Spt5 CTD code

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• mRNA capping enzymes interact with RNA polymerase II (Pol2) and Spt5 carboxy-terminal domains (CTDs) to target nascent transcripts.
• Fission yeast RNA triphosphatase Pct1 recognizes the Spt5 CTD, which contains a tandem repeat of the nonapeptide motif TPAWNSGSK.

Purpose of the Study:

• To determine the crystal structure of the Pct1·Spt5-CTD complex.
• To elucidate the molecular basis of Pct1 binding to the Spt5 CTD.
• To investigate the role of threonine phosphorylation in regulating Pct1-Spt5 CTD interactions.

Main Methods:

• X-ray crystallography to determine the structure of the Pct1·Spt5-CTD complex.
• Biochemical assays to assess the effect of threonine phosphorylation on binding.

Main Results:

• The crystal structure revealed two CTD docking sites on the Pct1 homodimer that bind TPAWN segments of the Spt5 CTD.
• Pct1 binding is dominated by van der Waals contacts with the tryptophan residue of the Spt5 CTD.
• The Spt5 CTD adopts a U-turn conformation upon binding to Pct1.
• Threonine phosphorylation of the Spt5 CTD antagonizes Pct1 binding.

Conclusions:

• Pct1 utilizes specific tryptophan interactions within the Spt5 CTD for binding.
• The Spt5 CTD is structurally plastic and its conformation is influenced by interacting proteins.
• Threonine phosphorylation acts as a binary switch regulating the interaction between Spt5 CTD and its receptors, contributing to an "Spt5 CTD code".