title: In vivo transcriptional analysis of the spliced leader RNA gene in the trypanosomatid Leptomonas seymouri.
abstract: Gene expression in all organisms requires the direct and indirect interaction of multiple proteins with specific DNA sequence elements. Using the monogenetic trypanosomatid, Leptomonas seymouri, we investigated the cis- and trans-acting components that determine expression of a central trypanosomatid RNA, the spliced leader (SL) RNA. Using base substitution mutagenesis and DNA transfection assays, we determined that the SL RNA gene promoter lies exclusively up-stream from the transcription initiation site. Accordingly, the SL RNA gene can be used as a gene cassette to express short heterologous RNAs of interest. We utilized two pharmacological agents, alpha-amanitin and tagetitoxin, and the detergent sarkosyl to assess components of the trans-acting machinery involved in transcription. The SL RNA inhibition pattern was distinct from that of alpha-tubulin, tRNA or ribosomal RNA. Taken together, these data suggest that the upstream SL RNA gene promoter serves to nucleate a transcriptional complex that is distinct, in either its initiation and/or elongation abilities, from other genes. A comparison of trypanosomatid SL RNA gene promoter structures with that found in the nematode Ascaris lumbricoides underscores a taxonomic difference in promoter architectures which may reflect differential requirements for the SL RNA in these organisms.
authors.affiliation: Department of Microbiology and Molecular Genetics, UMDNJ-Graduate School of Biomedical Sciences, Newark, NJ 07103, USA.
authors.forename: K
authors.initials: K
authors.lastname: Crenshaw-Williams
authors.forename: V
authors.initials: V
authors.lastname: Bellofatto
mesh_terms: D000818:Animals; D012321:DNA-Directed RNA Polymerases; D015870:Gene Expression; D017125:Genes, Protozoan; D011401:Promoter Regions, Genetic; D016053:RNA, Protozoan; D020038:RNA, Spliced Leader; D014158:Transcription, Genetic; D014351:Trypanosomatina / Q000235:genetics*
chemical_list: D016053:RNA, Protozoan; D020038:RNA, Spliced Leader; D012321:DNA-Directed RNA Polymerases