Biochemical characterization of Trypanosoma brucei RNA polymerase II.
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 alpha-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 CX(2)CX(10-15)CX(2)C 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.
Das
Anish
A
Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, 225 Warren Street, Newark, NJ 07103, USA.
Li
Hong
H
Liu
Tong
T
Bellofatto
Vivian
V
D000546:Amanitins / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D008018:Life Cycle Stages; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D020038:RNA, Spliced Leader; D016415:Sequence Alignment; D035581:Transcription Factor TFIIB / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000254:growth & development
D000546:Amanitins; D015800:Protozoan Proteins; D020038:RNA, Spliced Leader; D035581:Transcription Factor TFIIB; D012319:RNA Polymerase II