Single nucleotide resolution of promoter activity and protein binding for the Leishmania tarentolae spliced leader RNA gene

Abstract

In Kinetoplastid protozoa, trans-splicing is a central step in the maturation of nuclear mRNAs. In Leishmania, a common 39 nt spliced-leader (SL) is transferred via trans-splicing from the precursor 96 nt SL RNA to the 5′ terminus of all known protein-encoding RNAs. In this study, promoter elements of the L. tarentolae SL RNA gene have been identified with respect to transcriptional activity and putative transcription factor binding. We have mapped the essential regions in the SL RNA gene promoter at single nucleotide resolution using both in vivo transcription and in vitro protein/DNA binding approaches. Two regions located upstream of the SL RNA gene were identified: a GN 3CCC element at −39 to −33 and a GACN 5G element at −66 to −58 were essential for SL RNA gene transcription in stably transfected cells. Consistent with other known bipartite promoter elements, the spacing between the GN 3CCC and GACN 5G elements was found to be critical for proper promoter function and correct transcription start point selection, as was the distance between the two elements and the wild-type transcription start point. The GACN 5G element interacts specifically and in a double-stranded form with a protein(s) in Leishmania nuclear extracts. The degree of this protein–DNA interaction in vitro correlated with SL RNA gene transcription efficiency in vivo, consistent with a role of the protein as a transcription factor. The core nucleotides GACN 5G fit the consensus PSE promoter structure of pol II-transcribed snRNA genes in metazoa.