Protein coding transcription and control of vsp gene expression in the protozoan parasite Giardia lamblia

Abstract

Giardia lamblia is an early branching eukaryote and although distinctly eukaryotic in its cell and molecular biology, transcription in G. lamblia demonstrates important differences from these processes in higher eukaryotes. α-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 α-amanitin and found that unlike most other eukaryotes, RNAP II transcription in Giardia is resistant to 1 mg/ml amanitin. To better understand transcription in G. lamblia, we identified 10 out of the 12 known eukaryotic rph genes, including all ten that are required for viability in Saccharomyces cerevisiae. The amanitin motif (amanitin binding site) of Rpbl from G. lamblia differs from other eukaryotes at six highly conserved sites in which substitutions have been associated with amanitin resistance in other organisms. These observations of amanitin resistance provide a molecular framework for the development of novel drugs with selective activity against G. lamblia. Giardia trophozoites exhibit antigenic variation of a surface protein encoded by a family of genes known as the vsp genes. A single trophozoite expresses only one vsp at a time and it has been previously determined that steady state mRNA of only the expressed vsp is detectable in Northern blots. Our nuclear run-on assays indicated transcription of only the expressed vsp genes, suggesting that control is primarily at the level of transcription rather than post-transcription. In order to better understand vsp gene control mechanisms, we used a luciferase reporter to determine the vsp core promoter, which is present within 100 bp upstream of the ORF in the case of vspCS and vspA6. The fact that the vsp promoter is able to drive expression irrespective of the antigenic variant type indicates that control requires a chromosomal context as do most epigenetic mechanisms of control. Based on the existing data, we provide a privileged site model for the control of vsp gene expression, in which vsp transcription takes place in a set nuclear location which is occupied by a singe vsp locus at a time.