Scimago Lab
powered by Scopus
call: +1.631.470.9640
Mon-Fri 10 am - 2 pm EST


Medical Science Monitor Basic Research


eISSN: 1643-3750

Get your full text copy in PDF

Carboxy terminal domain of the largest subunit of RNA polymerase II of Leishmania donovani has an unusually low number of phosphorylation sites.

Shalini Sharma, Aditi Das, Arindam Dasgupta, Dwijen Sarkar, Hemanta Majumder

Med Sci Monit 2002; 8(5): CR341-350

ID: 420838

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.

This paper has been published under Creative Common Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) allowing to download articles and share them with others as long as they credit the authors and the publisher, but without permission to change them in any way or use them commercially.
I agree