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J. Biol. Chem., Vol. 278, Issue 30, 27804-27810, July 25, 2003

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Giardia lamblia RNA Polymerase II

AMANITIN-RESISTANT TRANSCRIPTION^*

Vishwas Seshadri , Andrew G. McArthur , Mitchell L. Sogin  and Rodney D. Adam  ¶ ||

From the Departments of Microbiology and Immunology and ^¶Medicine, University of Arizona College of Medicine, Tucson, Arizona 85724-5049 and Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, Massachusetts 02543-1015

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 -amanitin and found that unlike most other eukaryotes, RNAP II transcription in Giardia is resistant to 1 mg/ml amanitin. In contrast, 50 µg/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.

Received for publication, March 31, 2003 , and in revised form, May 3, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF510641–51 (rpb genes) and AY245002 (leucyl-tRNA gene).

^* This work was supported in part by National Institutes of Health Grants AI43273 (to M. L. S.) and AI51089 (to A. G. M.). Additional support was provided by the G. Unger Vetlesen Foundation and LI-COR Biotechnology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. of Microbiology/Immunology, University of Arizona, 1501 N. Campbell, Tucson, AZ 85724-5049. Tel.: 520-626-6430; Fax: 626-2100; E-mail: adamr{at}u.arizona.edu.

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