In vitro transcription of mutated Leishmania tarentolae spliced leader RNAgenes approximates in vivo patterns

To elucidate the process of transcription in the kinetoplastids and to aid in the purification of transcription factors, we have developed a transcrip tionally-competent nuclear extract from Leishmania tarentolae for the study of the spliced leader (SL) RNA gene. The extract was competent to transcri be a tagged SL RNA gene. The in vitro SL RNA transcripts initiated accurate ly and their synthesis was dependent on the presence of the promoter define d in vivo. The nuclear extract was then challenged rigorously using an exha ustive set of mutated SL RNA gene templates previously tested for transcrip tional activity in vivo. Mutation of four nucleotides (CCGG) at positions - 34 to -31 had a detrimental effect on transcription in vitro; the CC dinucl eotide overlaps one element necessary in vivo. Similarly, four nucleotides (TGAC; positions -67 to -64) important for transcription in vitro overlappe d the other core promoter element defined in vivo, but were generally not e ffective as point mutations. The promoter-binding ability of the transcript ionally-competent extract for the -60 region mutations mirrored the in vitr o transcription pattern. Although it does not reflect precisely the in vivo results, this in vitro system provides us with an important tool for monit oring the purification of potential transcription factors, as well as the b asis for future reconstitution experiments. (C) 2000 Elsevier Science B.V. All rights reserved.