Characterization of Toxoplasma gondii Spt5 like transcription elongation factor

Elongation has emerged as a highly regulated step in the multistage process of transcription. Control of gene expression mediated through transcription elongation remains an unexplored area of study in Toxoplasma gondii where the demands of complex lifecycle necessitate a regulated transcription program. Here, we elucidate the central role of Spt5 homolog in T. gondii mRNA transcription. We demonstrate that TgSpt5 functions in conjunction with a small zinc finger protein TgSpt4. TgSpt5 interacts with TgRpb1, the largest subunit of RNA polymerase II and associates with actively transcribed genes. Enrichment of TgSpt5 towards the 3' end of genes coinciding with P-Ser2 form of RNAPII, a marker of active elongation further underscores its pivotal role in transcription. TgSpt5 undergoes phosphorylation mediated through Toxoplasma Cdk9 homolog, TgCrk9, which appears crucial for its function. Inhibition of TgCrk9, which also regulates RNAPII by differential phosphorylation of its C terminal domain, results in loss of TgSpt5 enrichment at 3' sites of the genes and an overall repressive effect on parasite progression. TgSpt5 along with TgSpt4 could successfully complement the loss of function mutations in yeast counterparts emphasizing its functional significance. Together, the results highlight the possible role of TgSpt5 in transcript elongation regulated through phosphorylation by TgCrk9.