A conserved upstream element is essential for transcription of the Leishmania tarentolae mini-exon gene

EMBO J. 1994 Nov 15;13(22):5460-9.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Exons / genetics*
  • Gene Expression Regulation*
  • Genes, Synthetic
  • Genetic Vectors
  • Leishmania / genetics*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Promoter Regions, Genetic*
  • RNA Caps / genetics
  • RNA Polymerase II / metabolism*
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • RNA, Protozoan / biosynthesis
  • RNA, Protozoan / genetics
  • Repetitive Sequences, Nucleic Acid
  • Transcription, Genetic*

Substances

  • RNA Caps
  • RNA, Messenger
  • RNA, Protozoan
  • RNA Polymerase II