Known transcription factors of trypanosomatid organisms are extremely divergent in amino acid sequence to their counterparts in other eukaryotes. Sequence similarity is so limited that factors have been primarily identified by functional and structural studies. In addition, trypanosomatids may have evolved factors that are specific to this group of organisms. Under these circumstances, an in vitro transcription system is invaluable as it allows for unambiguous determination of a factor's transcriptional role. Here we describe procedures for the preparation of transcriptionally active extracts, detail in vitro transcription reactions, and specify the particular strategy necessary to detect template-derived RNA in this system. As examples of how to use this system, we describe factor depletion from extract and antibody-mediated interference with a factor's transcriptional function. Furthermore, we detail a promoter pull-down assay that makes use of the extracts and facilitates analysis of a factor's interaction with promoter DNA.
Keywords: Extract preparation; In vitro transcription; Primer extension; Promoter pull-down assay; Radiolabeling of DNA.