Active RNA Polymerase I of Trypanosoma brucei Harbors a Novel Subunit Essential for Transcription
- 1 September 2007
- journal article
- Published by Taylor & Francis Ltd in Molecular and Cellular Biology
- Vol. 27 (17), 6254-6263
- https://doi.org/10.1128/mcb.00382-07
Abstract
A unique characteristic of the protistan parasite Trypanosoma brucei is a multifunctional RNA polymerase I which, in addition to synthesizing rRNA as in other eukaryotes, transcribes gene units encoding the major cell surface antigens variant surface glycoprotein and procyclin. Thus far, purification of this enzyme has revealed nine orthologues of known subunits but no active enzyme. Here, we have epitope tagged the specific subunit RPB6z and tandem affinity purified RNA polymerase I from crude extract. The purified enzyme was active in both a nonspecific and a promoter-dependent transcription assay and exhibited enriched protein bands with apparent sizes of 31, 29, and 27 kDa. p31 and its trypanosomatid orthologues were identified, but their amino acid sequences have no similarity to proteins of other eukaryotes, nor do they contain a conserved sequence motif. Nevertheless, p31 cosedimented with purified RNA polymerase I, and RNA interferance-mediated silencing of p31 was lethal, affecting the abundance of rRNA. Moreover, extract of p31-silenced cells exhibited a specific defect in transcription of class I templates, which was remedied by the addition of purified RNA polymerase I, and an anti-p31 serum completely blocked RNA polymerase I-mediated transcription. We therefore dubbed this novel functional component of T. brucei RNA polymerase I TbRPA31.Keywords
This publication has 43 references indexed in Scilit:
- Spliced Leader RNA Gene Transcription in Trypanosoma brucei Requires Transcription Factor TFIIHEukaryotic Cell, 2007
- Diversification of Function by Different Isoforms of Conventionally Shared RNA Polymerase SubunitsMolecular Biology of the Cell, 2007
- Regulation of antigen gene expression in Trypanosoma bruceiTrends in Parasitology, 2005
- Highly Efficient Tandem Affinity Purification of Trypanosome Protein Complexes Based on a Novel Epitope CombinationEukaryotic Cell, 2005
- Characterization of a Multisubunit Transcription Factor Complex Essential for Spliced-Leader RNA Gene Transcription in Trypanosoma bruceiMolecular and Cellular Biology, 2005
- The Genome of the African Trypanosome Trypanosoma bruceiScience, 2005
- A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma bruceiNature, 2001
- Inhibition of Trypanosoma brucei Gene Expression by RNA Interference Using an Integratable Vector with Opposing T7 PromotersPublished by Elsevier BV ,2000
- Efficient production of functional mRNA mediated by RNA polymerase I in Trypanosoma bruceiNature, 1991
- Complete sequence of the gene encoding the largest subunit of RNA polymerase I of Trypanosoma bruceiFEBS Letters, 1989