Biochemical characterization of Trypanosoma brucei RNA polymerase II

Anish Das, Hong Li, Tong Liu, Vivian Bellofatto

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

In Trypanosoma brucei, transcription by RNA polymerase II accounts for the expression of the spliced leader (SL) RNA and most protein coding mRNAs. To understand the regulation of RNA polymerase II transcription in these parasites, we have purified a transcriptionally active enzyme through affinity chromatography of its essential subunit, RPB4. The enzyme preparation is active in both promoter-independent and promoter-dependent in vitro transcription assays. Importantly, the enzyme is sensitive to α-amanitin inhibition, a hallmark of eukaryotic RNA polymerase II enzymes. Using mass spectrometric analysis we have identified the previously unobserved RPB12 subunit of T. brucei RNA polymerase II. TbRPB12 contains a conserved CX2CX10-15CX2C zinc binding motif that is characteristic of other eukaryotic RPB12 polypeptides. We also identified seven proteins that associate with T. brucei RNA polymerase II. While both bioinformatics and biochemical analysis have focused on the subunit structure of trypanosome RNA polymerases, this is the first study that reveals a functional RNA polymerase II enzyme.

Original languageEnglish (US)
Pages (from-to)201-210
Number of pages10
JournalMolecular and Biochemical Parasitology
Volume150
Issue number2
DOIs
StatePublished - Dec 2006

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

Keywords

  • Gene regulation
  • Proteomics
  • RNA polymerase II
  • Transcription
  • Trypanosoma brucei

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