Active RNA Polymerase I of Trypanosoma brucei Harbors a Novel Subunit Essential for Transcription

Abstract

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.