In vivo transcriptional analysis of the spliced leader RNA gene in the trypanosomatid Leptomonas seymouri.
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.
Crenshaw-Williams
K
K
Department of Microbiology and Molecular Genetics, UMDNJ-Graduate School of Biomedical Sciences, Newark, NJ 07103, USA.
Bellofatto
V
V
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*
D016053:RNA, Protozoan; D020038:RNA, Spliced Leader; D012321:DNA-Directed RNA Polymerases