Carboxy terminal domain of the largest subunit of RNA polymerase II of Leishmania donovani has an unusually low number of phosphorylation sites.
BACKGROUND
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II in higher eukaryotes has an altered form in Leishmania donovani. To determine whether this is a general feature 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 discreteness observed may be due to a functional constraint delineating parasite from host.


METHODS
The gene for L. donovani rpolIILS was picked up and sequenced. The CTD of L. donovani rpolIILS was purified as 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 was done using anti-CTD antibody.


RESULTS
The L. donovani rpolIILS is encoded by a single-copy gene. Its transcript is matured postranscriptionally, with the mini-exon trans-spliced 397 bases upstream of the initiation site. The uniqueness of Leishmania rpolIILS CTD according to prediction analysis was corroborated with in vitro phosphorylation of the recombinant protein. Photoaffinity labelling of L. donovani nuclear run-on transcripts and immunoblot analysis using anti-CTD antibody could identify the active form of RNA polymerase II enzyme in this parasite.


CONCLUSIONS
The L. donovani rpolIILS possesses a unique C-terminal extension lacking the characteristic repeats but containing serine residues as a potential phosphorylation site. Anti-CTD antibody could recognize a single molecular species for the RNA pol II enzyme in L. donovani.
Dasgupta
Arindam
A
Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, USA.
Sharma
Shalini
S
Das
Aditi
A
Sarkar
Dwijen
D
Majumder
Hemanta
H
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D005091:Exons; D015723:Gene Library; D015151:Immunoblotting; D016830:Kinetoplastida / Q000502:physiology; D007893:Leishmania donovani / Q000201:enzymology*; D009693:Nucleic Acid Hybridization; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry*; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D012333:RNA, Messenger; D011994:Recombinant Proteins; D012313:RNA; D012319:RNA Polymerase II