Structural and functional characterization of the Leishmania amazonensis ribosomal RNA promoter.

The promoter region of the ribosomal RNA (rRNA) genes of Leishmania amazonensis was characterised and the transcription start point, defined by primer extension, was shown to be a T residue, 1048 nucleotides upstream of the beginning of the 18S sequence. A repetitive element of 60 bp was identified in the intergenic spacer. This element did not show sequence similarity with the region around the transcription start point. Conserved sequences were found in the external transcribed spacer of L. amazonensis, Trypanosoma cruzi and Crithidia fasciculata rRNA genes, 150 nucleotides downstream of the transcription start point. These sequences might be involved in processing events of the rRNA precursor molecule. The general organisation of the gene resembles the pattern observed for the ribosomal cistron in eukaryotic cells. Constructs containing the L. amazonensis promoter region upstream of the chloramphenicol acetyltransferase (cat) gene were able to drive the expression of the reporter gene in transient transfection experiments. CAT expression could be detected even when no trans-splicing acceptor sequence was added to the constructs, although its presence enhanced 5-fold the level of CAT activity. Species-specificity of the RNA polymerase I promoter activity was also demonstrated since constructs containing the L. amazonensis promoter region were unable to drive CAT expression when transfected into the related trypanosomatids, T. cruzi, C. fasciculata and Endotrypanum schaudini.