Analysis of ribosomal RNA transcription termination and 3′ end processing in Leishmania amazonensis

The control of gene expression in the human parasite Leishmania occurs mainly at the post-transcriptional level. Nevertheless, basic cell processes such as ribosome biogenesis seem to be conserved. Mature ribosomal RNAs (rRNAs) are synthesized from typical RNA polymerase I (Pol I) promoters and processed by pathways analogous to other eukaryotes. To further understand Pol I transcription control in these parasites, we have analyzed transcription termination and processing of the rDNA in Leishmania amazonensis. 3′-end S1 mapping of rRNA precursors identified three termini, one corresponding to the mature 28S rRNA and two to the rDNA intergenic spacer (IGS), termed T1 and T2, for precursors which are 185 and 576 nucleotides longer, respectively. Both T1 and T2 are associated with conserved G + C rich elements that have the potential to form hairpin structures and T-rich clusters. We found that two fragments of 423 bp and 233 bp, flanking sites T1 and T2 respectively when placed upstream of the green fluorescent protein gene (GFP), negatively affected the Pol I-driven transcription of this gene, which suggests the presence of a transcription terminator element in these regions. Deletion analysis pointed to a CCCTTTT heptamer as part of the putative terminator and suggested that the hairpins are processing signals.