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

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

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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 (rpol IILS) and analyzed its C-terminal domain. The discreteness observed may be due to a functional constraint delineating parasite from host. Material/Methods: The gene for L. donovani rpol IILS 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 rpol IILS 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 rpol IILS 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. Conclusion: The L. donovani rpol IILS 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.

Original languageEnglish (US)
JournalMedical Science Monitor
Volume8
Issue number5
StatePublished - 2002
Externally publishedYes

Fingerprint

Leishmania donovani
RNA Polymerase II
Recombinant Proteins
Antibodies
Parasites
Kinetoplastida
Phosphorylation
Genes
RNA polymerase II largest subunit
Leishmania
Enzymes
Eukaryota
Complex Mixtures
Serine
Exons
Phosphotransferases

Keywords

  • C-terminal domain
  • Cloning
  • Leishmania donovani
  • Phosphorylation
  • RNA polymerase II largest subunit

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Carboxy terminal domain of the largest subunit of RNA polymerase II of Leishmania donovani has an unusually low number of phophorylation sites. / Dasgupta, Arindam; Sharma, Shalini; Das, Aditi; Sarkar, Dwijen; Majumder, Hemanta K.

In: Medical Science Monitor, Vol. 8, No. 5, 2002.

Research output: Contribution to journalArticle

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T1 - Carboxy terminal domain of the largest subunit of RNA polymerase II of Leishmania donovani has an unusually low number of phophorylation sites

AU - Dasgupta, Arindam

AU - Sharma, Shalini

AU - Das, Aditi

AU - Sarkar, Dwijen

AU - Majumder, Hemanta K.

PY - 2002

Y1 - 2002

N2 - 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 (rpol IILS) and analyzed its C-terminal domain. The discreteness observed may be due to a functional constraint delineating parasite from host. Material/Methods: The gene for L. donovani rpol IILS 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 rpol IILS 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 rpol IILS 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. Conclusion: The L. donovani rpol IILS 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.

AB - 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 (rpol IILS) and analyzed its C-terminal domain. The discreteness observed may be due to a functional constraint delineating parasite from host. Material/Methods: The gene for L. donovani rpol IILS 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 rpol IILS 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 rpol IILS 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. Conclusion: The L. donovani rpol IILS 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.

KW - C-terminal domain

KW - Cloning

KW - Leishmania donovani

KW - Phosphorylation

KW - RNA polymerase II largest subunit

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