共 44 条
Purification of an eight subunit RNA polymerase I complex in Trypanosoma brucei
被引:33
作者:
Nguyen, Tu N.
Schimanski, Bernd
Zahn, Andre
Klumpp, Birgit
Guenzl, Arthur
机构:
[1] Univ Connecticut, Ctr Hlth, Dept Genet & Dev Biol, Farmington, CT 06030 USA
[2] Univ Connecticut, Ctr Hlth, Dept Mol Microbial & Struct Biol, Farmington, CT 06030 USA
[3] Univ Tubingen, Med Naturwissen Forschungzentrum, D-72074 Tubingen, Germany
关键词:
RNA polymerase I;
T brucei;
RPB6;
RPB8;
RPB10;
RPC19;
D O I:
10.1016/j.molbiopara.2006.02.023
中图分类号:
Q5 [生物化学];
Q7 [分子生物学];
学科分类号:
071010 ;
081704 ;
摘要:
Trypanosoma brucei harbors a unique multifunctional RNA polymerase (pol) I which transcribes, in addition to ribosomal RNA genes, the gene units encoding the major cell surface antigens variant surface glycoprotein and procyclin. In consequence, this RNA pol I is recruited to three structurally different types of promoters and sequestered to two distinct nuclear locations, namely the nucleolus and the expression site body. This versatility may require parasite-specific protein-protein interactions, subunits or subunit domains. Thus far, data mining of trypanosomatid genomes have revealed 13 potential RNA pol I subunits which include two paralogous sets of RPB5, RPB6, and RPB10. Here, we analyzed a cDNA library prepared from procyclic insect form T brucei and found that all 13 candidate subunits are co-expressed. Moreover, we PTP-tagged the largest subunit TbRPA1, tandem affinity-purified the enzyme complex to homogeneity, and determined its subunit composition. In addition to the already known subunits RPAI, RPA2, RPC40, 1RPB5, and RPA12, the complex contained RPC19, RPB8, and 1RPB10. Finally, to evaluate the absence of RPB6 in our purifications, we used a combination of epitope-tagging and reciprocal coimmunoprecipitation to demonstrate that 1RPB6 but not 2RPB6 binds to RNA pol I albeit in an unstable manner. Collectively, our data strongly suggest that T. brucei RNA pol I binds a distinct set of the RPB5, RPB6, and RPB10 paralogs. (c) 2006 Elsevier B.V. All rights reserved.
引用
收藏
页码:27 / 37
页数:11
相关论文