Giardia lamblia RNA polymerase II: amanitin-resistant transcription.

ABSTRACT

Giardia lamblia is an early branching eukaryote, and although distinctly eukaryotic in its cell and molecular biology, transcription and translation in G. lamblia demonstrate important differences from these processes in higher eukaryotes. The cyclic octapeptide amanitin is a relatively selective inhibitor of eukaryotic RNA polymerase II (RNAP II) and is commonly used to study RNAP II transcription. Therefore, we measured the sensitivity of G. lamblia RNAP II transcription to alpha-amanitin and found that unlike most other eukaryotes, RNAP II transcription in Giardia is resistant to 1 mg/ml amanitin. In contrast, 50 microg/ml amanitin inhibits 85% of RNAP III transcription activity using leucyl-tRNA as a template. To better understand transcription in G. lamblia, we identified 10 of the 12 known eukaryotic rpb subunits, including all 10 subunits that are required for viability in Saccharomyces cerevisiae. The amanitin motif (amanitin binding site) of Rpb1 from G. lamblia has amino acid substitutions at six highly conserved sites that have been associated with amanitin resistance in other organisms. These observations of amanitin resistance of Giardia RNA polymerase II support previous proposals of the mechanism of amanitin resistance in other organisms and provide a molecular framework for the development of novel drugs with selective activity against G. lamblia.