A conserved upstream element is essential for transcription of the Leishmania tarentolae mini-exon gene.
We demonstrate that the mini-exon genes of Leishmania tarentolae are individually transcribed by an enzyme pharmacologically identified as RNA polymerase II. To study transcription in these ancient eukaryotes, a stable transformation assay using an episomal mini-exon gene was developed. The introduced mini-exon gene, which had been marked with a 40 bp tag, yielded the predicted tagged transcript. An upstream cis-acting element that was essential for transcription of the mini-exon gene was identified by site-directed mutagenesis. Block substitution mutagenesis of the -1 to +9 and +10 to +19 regions of the exon results in 20- to 100-fold decreased levels of the tagged transcript in steady-state RNA. However, since these two mutations resulted in only a 2- to 3-fold decrease in nascent RNA levels, steady-state levels appear to be affected greatest by the stability of the resulting transcript. In contrast, mutation of the -67/-58 region resulted in undetectable levels of both steady-state and nascent RNA from the introduced gene. We conclude, therefore, that this upstream element, which is highly conserved in all Leishmania species, is a component of the mini-exon gene promoter.
Saito
R M
RM
Department of Microbiology and Immunology, University of California, Los Angeles, CA 90024.
Elgort
M G
MG
Campbell
D A
DA
D000818:Animals; D001483:Base Sequence; D005091:Exons / Q000235:genetics*; D005786:Gene Expression Regulation; D005813:Genes, Synthetic; D005822:Genetic Vectors; D007891:Leishmania / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012315:RNA Caps / Q000235:genetics; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D016053:RNA, Protozoan / Q000096:biosynthesis / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic
D012315:RNA Caps; D012333:RNA, Messenger; D016053:RNA, Protozoan; D012319:RNA Polymerase II