Analysis of the 5S rRNA gene promoter from Acanthamoeba castellanii

Abstract

The promoter region of the Acanthamoeba 5S rRNA gene was analysed by in vitro transcription of several 5' and 3' deletion and substitution mutants, as well as a series of linker scanning mutants. The promoter consists of three sequence regions contained entirely within the gene; two of these correspond to the A and C boxes that bind TFIIIA, found in the genes from other genera. In addition, a region immediately 3' to the transcription start site has a strong effect on initiation efficiency. No strict requirement was found for specific sequences 5' to the transcription start site. Substitution of a consensus TATA box at -29 had only a modest effect on transcription, and deletion or substitution of sequences between -15 and -10 as well as -34 and -21 was only modestly more active than the wild-type template. Analysis of 3' deletions sets the 3' end-point of the promoter between +79 and +97, and demonstrates the importance of a T-rich region in transcription termination. Taken together, these results suggest that promoter elements within the Acanthamoeba 5S RNA gene are somewhat redundant, with the exception of a sequence between +50 and +60, which functions in binding TFIIIA. Remarkably, polymerase chain reaction product templates containing only non-specific 5' ends between -6 and +1 relative to the transcription start site are fully functional, demonstrating that no external DNA scaffold is needed for TFIIIB and RNA polymerase III binding, and that productive initiation can be mediated solely by protein-DNA interactions within the coding region of the 5S gene.