RESUMEN
In the protozoan parasite Trypanosoma brucei, the two main surface glycoprotein genes are transcribed by RNA polymerase I (pol I) instead of RNA pol II, the polymerase committed to the production of mRNA in eukaryotes. This unusual feature might be accomplished by the recruitment of specific subunits or cofactors that allow pol I to transcribe protein-coding RNAs. Here, we report that transcription mediated by pol I requires TbRPB7, a dissociable subunit of the pol II complex. TbRPB7 was found to interact with two pol I-specific subunits, TbRPA1 and TbRPB6z. Pol I-specific transcription was affected on depletion of TbRPB7 in run-on assays, whereas recombinant TbRPB7 increased transcription driven by a pol I promoter. These results represent a unique example of a functional RNA polymerase chimaera consisting of a core pol I complex that recruits a specific pol II subunit.
Asunto(s)
Subunidades de Proteína/metabolismo , Proteínas Protozoarias/metabolismo , ARN Polimerasa II/metabolismo , ARN Polimerasa I/metabolismo , Transcripción Genética , Trypanosoma brucei brucei/enzimología , Animales , Núcleo Celular/metabolismo , Genes Reporteros , Subunidades de Proteína/genética , Proteínas Protozoarias/genética , Interferencia de ARN , ARN Polimerasa I/genética , ARN Polimerasa II/química , ARN Polimerasa II/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/genéticaRESUMEN
We set out to find the "fenestrin" gene, a gene whose protein is associated with numerous cellular apertures, including the nuclear exchange junction in mating Tetrahymena thermophila. First we developed protocols for imaging and isolating intact nuclear exchange junctions from conjugating cells. Proteins from these junctions were purified using SDS-PAGE, subjected to limited proteolysis, and precise molecular weights were determined by mass spectrometry. Using Protein Prospector software and the published Tetrahymena Genome Database, genes for 15 of the most abundant proteins found in our extracts were identified. The most promising candidate was cloned by PCR, fused to yellow fluorescent protein (YFP), and placed under the control of an inducible metallothionein promoter. YFP-localization within live Tetrahymena transformants strongly suggested that one of these genes encoded the fenestrin protein, a result that was subsequently confirmed by Western blotting.
