Accurate transcription of the Trypanosoma brucei U2 small nuclear RNA gene in a homologous extract.

The Journal of Biological Chemistry
A GünzlE Ullu

Abstract

In vitro transcription systems are a classic means to dissect mechanisms of gene expression at the molecular level. To begin an analysis of the biochemistry of gene expression in trypanosomes, we established an in vitro transcription system from cultured insect forms of Trypanosoma brucei. As a model we used the U2 snRNA gene which in vivo is transcribed by an RNA polymerase with characteristics of animal RNA polymerase III. To obtain maximum sensitivity in our assay, we adapted the so-called G-less cassette approach to the U2 snRNA gene promoter. Since an intragenic control region is required for accurate expression in vivo, we generated a series of mutations to substitute all guanosine residues in the intragenic control region. These mutants were shown to retain full transcriptional activity in vivo after transient expression in insect-form trypanosomes. In a cell-free extract, synthesis of the U2 G-less cassette RNA is correctly initiated, is mediated by RNA polymerase III as determined by RNA polymerase inhibitor studies, and is dependent on the integrity of the upstream B box element.

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Citations

Nov 19, 2003·Microbes and Infection·David A CampbellNancy R Sturm
Sep 1, 1997·Molecular and Biochemical Parasitology·V NakaarC Tschudi
Mar 13, 1998·Molecular and Biochemical Parasitology·J L HuieV Bellofatto
Aug 27, 1998·Molecular and Biochemical Parasitology·Y Tzfati, J Shlomai
Jan 1, 1997·Annual Review of Microbiology·M G Lee, L H Van der Ploeg
Feb 20, 2010·Journal of Biomedicine & Biotechnology·Santiago Martínez-CalvilloElisa E Figueroa-Angulo
Mar 17, 1999·Proceedings of the National Academy of Sciences of the United States of America·J E DonelsonN M El-Sayed
Mar 6, 2010·Journal of Biomedicine & Biotechnology·Consuelo GomezMario A Rodríguez
Oct 24, 2007·Molecular and Biochemical Parasitology·Jia-Peng RuanChristian Tschudi
Feb 13, 2007·Memórias do Instituto Oswaldo Cruz·D L AmbrósioR M B Cicarelli
Dec 31, 2014·Eukaryotic Cell·Norma E Padilla-MejíaSantiago Martínez-Calvillo
Jan 16, 1999·Molecular and Cellular Biology·N R SturmD A Campbell

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