The role of Toxoplasma TFIIS-like protein in the early stages of mRNA transcription

• Published in: Biochimica et biophysica acta. General subjects Vol. 1866; no. 12; p. 130240
• Main Authors: Mitra, Pallabi, Banerjee, Sneha, Khandavalli, Chittiraju, Deshmukh, Abhijit S.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.12.2022
• Subjects: confocal microscopy; DNA; DNA-directed RNA polymerase; eukaryotic cells; gene expression; gene expression regulation; genes; precipitin tests; promoter regions; Protozoa; RNAPII transcription; TFIIS; Toxoplasma; Toxoplasma gondii; Transcription elongation; Transcription initiation; yeasts; zinc finger motif; Toxoplasma; TFIIS; Transcription elongation; RNAPII transcription; Transcription initiation
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2022.130240

The mRNA transcription is a multistep process involving distinct sets of proteins associated with RNA polymerase II (RNAPII) through various stages. Recent studies have highlighted the role of RNAPII-associated proteins in facilitating the assembly of functional complexes in a crowded nuclear milieu. RNAPII dynamics and gene expression regulation have been primarily studied in model eukaryotes like yeasts and mammals and remain largely unchartered in protozoan parasites like Toxoplasma gondii, where considerable gene expression changes accompany stage differentiations. Here we report a key modulator of RNAPII activity, TFIIS in Toxoplasma gondii (TgTFIIS). A Pull-down assay demonstrated that TgTFIIS binds to RNAPII subunit TgRPB1. Truncation mutants of TFIIS help us define the regions critical for its binding to TgRPB1. Co-immunoprecipitation analysis confirmed the interaction between the native TgTFIIS and TgRPB1. Confocal microscopy revealed a predominantly nuclear localization. Native TgTFIIS was able to bind promoter DNA which was consistent with the CHIP results. TgTFIIS complements initiation defects in yeast mutants, and the regions implicated in RNAPII binding appeared essential for this function. Interestingly, the C-terminal zinc finger domain necessary for its potential elongation function is dispensable for TgRPB1 binding. TgTFIIS was found to be associated with the promoter region along with its association with the ORF on an RNAPII transcribed gene. The observations were in line with the potential role of TgTFIIS in early events of RNAPII transcription in addition to elongation. The study elucidates the potential role of RNAPII-associated proteins in multiple steps of transcription. •Toxoplasma TFIIS(TgTFIIS) homolog binds the RPB1 subunit of RNAPII.•TgTFIIS complements yeast initiation defect.•Native TgTFIIS binds promoter DNA.•TgTFIIS occupies both the promoter region and gene body of an RNAPII transcribed gene.•TgTFIIS has a role in early events in RNAPII transcription besides elongation.