Abstract

BACKGROUND: The C-terminal domain (CTD) of the largest subunit of RNA polymeraseII in higher eukaryotes has an altered form in Leishmania donovani. To determine whether this is a generalfeature of the kinetoplastida and to investigate the role of this domain in parasitic RNA pol II transcription,we isolated the gene encoding RNA pol II LS (rpolIILS) and analyzed its C-terminal domain. The discretenessobserved may be due to a functional constraint delineating parasite from host. MATERIAL/METHODS: Thegene for L. donovani rpolIILS was picked up and sequenced. The CTD of L. donovani rpolIILS was purifiedas a His-tagged recombinant protein and phosphorylated with a crude kinase extract from L. donovani.An immunoblot analysis of the phosphorylated CTD and photo-crosslinked L. donovani nuclear extracts wasdone using anti-CTD antibody. RESULTS: The L. donovani rpolIILS is encoded by a single-copy gene. Itstranscript is matured postranscriptionally, with the mini-exon trans-spliced 397 bases upstream of theinitiation site. The uniqueness of Leishmania rpolIILS CTD according to prediction analysis was corroboratedwith in vitro phosphorylation of the recombinant protein. Photoaffinity labelling of L. donovani nuclearrun-on transcripts and immunoblot analysis using anti-CTD antibody could identify the active form ofRNA polymerase II enzyme in this parasite. CONCLUSIONS: The L. donovani rpolIILS possesses a unique C-terminalextension lacking the characteristic repeats but containing serine residues as a potential phosphorylationsite. Anti-CTD antibody could recognize a single molecular species for the RNA pol II enzyme in L. donovani.

Keywords: Cell Nucleus, Cloning, Molecular, Exons, Gene Library, Immunoblotting, Kinetoplastida, Leishmania donovani, Nucleic Acid Hybridization, Phosphorylation, Polymerase Chain Reaction, Protein Structure, Tertiary, RNA, RNA Polymerase II, RNA Processing, Post-Transcriptional, RNA Splicing, RNA, Messenger, Recombinant Proteins, Research Support, Non-U.S. Gov, Sequence Analysis, DNA, Transcription, Genetic, Ultraviolet Rays