Biochemical characterization of Trypanosoma brucei RNA polymerase II

TY - JOUR

T1 - Biochemical characterization of Trypanosoma brucei RNA polymerase II

AU - Das, Anish

AU - Li, Hong

AU - Liu, Tong

AU - Bellofatto, Vivian

N1 - Funding Information: This work was supported by NIH-NIAID grant AI 29478 to VB and NS046593 to HL. We thank Gina Conforti for the anti-TbRPB1 antibody. We thank Purnima Bhanot, David Lukac and Jennifer Palenchar for critical reading of this manuscript.

PY - 2006/12

Y1 - 2006/12

N2 - In Trypanosoma brucei, transcription by RNA polymerase II accounts for the expression of the spliced leader (SL) RNA and most protein coding mRNAs. To understand the regulation of RNA polymerase II transcription in these parasites, we have purified a transcriptionally active enzyme through affinity chromatography of its essential subunit, RPB4. The enzyme preparation is active in both promoter-independent and promoter-dependent in vitro transcription assays. Importantly, the enzyme is sensitive to α-amanitin inhibition, a hallmark of eukaryotic RNA polymerase II enzymes. Using mass spectrometric analysis we have identified the previously unobserved RPB12 subunit of T. brucei RNA polymerase II. TbRPB12 contains a conserved CX2CX10-15CX2C zinc binding motif that is characteristic of other eukaryotic RPB12 polypeptides. We also identified seven proteins that associate with T. brucei RNA polymerase II. While both bioinformatics and biochemical analysis have focused on the subunit structure of trypanosome RNA polymerases, this is the first study that reveals a functional RNA polymerase II enzyme.

AB - In Trypanosoma brucei, transcription by RNA polymerase II accounts for the expression of the spliced leader (SL) RNA and most protein coding mRNAs. To understand the regulation of RNA polymerase II transcription in these parasites, we have purified a transcriptionally active enzyme through affinity chromatography of its essential subunit, RPB4. The enzyme preparation is active in both promoter-independent and promoter-dependent in vitro transcription assays. Importantly, the enzyme is sensitive to α-amanitin inhibition, a hallmark of eukaryotic RNA polymerase II enzymes. Using mass spectrometric analysis we have identified the previously unobserved RPB12 subunit of T. brucei RNA polymerase II. TbRPB12 contains a conserved CX2CX10-15CX2C zinc binding motif that is characteristic of other eukaryotic RPB12 polypeptides. We also identified seven proteins that associate with T. brucei RNA polymerase II. While both bioinformatics and biochemical analysis have focused on the subunit structure of trypanosome RNA polymerases, this is the first study that reveals a functional RNA polymerase II enzyme.

KW - Gene regulation

KW - Proteomics

KW - RNA polymerase II

KW - Transcription

KW - Trypanosoma brucei

UR - http://www.scopus.com/inward/record.url?scp=33750607428&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33750607428&partnerID=8YFLogxK

U2 - https://doi.org/10.1016/j.molbiopara.2006.08.002

DO - https://doi.org/10.1016/j.molbiopara.2006.08.002

M3 - Article

C2 - 16962183

VL - 150

SP - 201

EP - 210

JO - Molecular and Biochemical Parasitology

JF - Molecular and Biochemical Parasitology

SN - 0166-6851

IS - 2

ER -