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

Abstract

To elucidate the process of transcription in the kinetoplastids and to aid in the purification of transcription factors, we have developed a transcriptionally-competent nuclear extract from Leishmania tarentolae for the study of the spliced leader (SL) RNA gene. The extract was competent to transcribe a tagged SL RNA gene. The in vitro SL RNA transcripts initiated accurately and their synthesis was dependent on the presence of the promoter defined in vivo. The nuclear extract was then challenged rigorously using an exhaustive set of mutated SL RNA gene templates previously tested for transcriptional activity in vivo. Mutation of four nucleotides (CCGG) at positions −34 to −31 had a detrimental effect on transcription in vitro; the CC dinucleotide overlaps one element necessary in vivo. Similarly, four nucleotides (TGAC; positions −67 to −64) important for transcription in vitro overlapped the other core promoter element defined in vivo, but were generally not effective as point mutations. The promoter-binding ability of the transcriptionally-competent extract for the −60 region mutations mirrored the in vitro transcription pattern. Although it does not reflect precisely the in vivo results, this in vitro system provides us with an important tool for monitoring the purification of potential transcription factors, as well as the basis for future reconstitution experiments.