SPT5

TgSpt5 is a transcription elongation factor in Toxoplasma gondii that couples RNA polymerase II to productive elongation (PMID:30707945). It forms a functional complex with the small zinc-finger protein TgSpt4 (PMID:30707945) and physically associates with TgRpb1, the largest subunit of RNAPII, directly linking it to the elongation machinery (PMID:30707945). By chromatin immunoprecipitation, TgSpt5 occupies actively transcribed genes and is enriched toward their 3' ends, coincident with the elongation-associated P-Ser2 form of RNAPII (PMID:30707945). Its chromatin engagement is controlled by phosphorylation: the Cdk9 homolog TgCrk9 phosphorylates TgSpt5, and TgCrk9 inhibition abolishes TgSpt5 enrichment at 3' gene sites and impairs parasite progression (PMID:30707945). The functional conservation of this activity is demonstrated by the ability of TgSpt5/TgSpt4 to complement loss of the yeast Spt4/Spt5 counterparts (PMID:30707945).

1. 2019 Medium

   Establishing whether Toxoplasma possesses a conserved Spt4/Spt5 elongation module addressed whether the parasite uses the canonical eukaryotic elongation apparatus; the finding defined TgSpt5 as part of a functional complex with the zinc-finger protein TgSpt4.

   Evidence Co-immunoprecipitation and functional interaction studies in T. gondii, with yeast complementation confirming conservation

   PMID:30707945

   Open questions at the time

   • Stoichiometry and structural architecture of the TgSpt4/TgSpt5 complex not resolved
   • Whether the complex has additional parasite-specific subunits unknown
2. 2019 Medium

   Determining how TgSpt5 connects to the transcription machinery showed it physically interacts with the RNAPII largest subunit TgRpb1, placing it directly on elongating polymerase.

   Evidence Co-immunoprecipitation/pulldown in T. gondii

   PMID:30707945

   Open questions at the time

   • Interaction from single Co-IP without reciprocal validation
   • Interface/domains mediating the TgRpb1 contact not mapped
3. 2019 Medium

   Mapping where TgSpt5 acts on genes established it as an in vivo elongation factor: it occupies actively transcribed genes and is enriched at 3' ends coincident with P-Ser2 RNAPII.

   Evidence Chromatin immunoprecipitation across multiple gene targets in T. gondii

   PMID:30707945

   Open questions at the time

   • Genome-wide occupancy profile not established
   • Direct effect on elongation rate or processivity not measured
4. 2019 Medium

   Identifying the regulatory input to TgSpt5 showed that the Cdk9 homolog TgCrk9 phosphorylates it, and this phosphorylation is required for TgSpt5 chromatin association and parasite growth.

   Evidence TgCrk9 kinase inhibition combined with ChIP and parasite growth assays in T. gondii

   PMID:30707945

   Open questions at the time

   • Specific phosphosites on TgSpt5 not identified
   • Whether TgCrk9 acts directly versus through an intermediate not fully distinguished

• How TgSpt5 phosphorylation state mechanistically couples to elongation rate, RNAPII pausing/release, and the broader parasite gene-expression program remains open.
• No structural model of the TgSpt4/TgSpt5-RNAPII assembly
• Direct measurement of elongation kinetics upon TgSpt5 depletion lacking
• Genome-wide targets and phosphosite-specific consequences uncharacterized