Identification of an essential proximal sequence element in the promoter of the telomerase RNA gene of Tetrahymena thermophila

Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes an d maintains telomeric DNA, Studies of telomeres and telomerase are facilita ted by the large number of linear DNA molecules found in ciliated protozoa, such as Tetrahymena thermophila. To examine the expression of telomerase, we investigated the transcription of the RNA polymerase Ill-directed gene e ncoding the RNA subunit (TER1) of this enzyme. A chimeric gene containing t he Glaucoma chattoni TER1 transcribed region flanked by 5' and 3' Tetrahyme na regions was used to identify promoter elements following transformation of Tetrahymena cells. Disruption of a conserved proximal sequence element ( PSE) located at -55 in the Tetrahymena TER1 5' flanking region eliminated e xpression of the chimeric gene. In addition, mutation of an A/T-rich elemen t at -25 decreased expression markedly. A gel mobility shift assay and prot ein-DNA cross-linking identified a PSE-binding polypeptide of 50-60 kDa in Tetrahymena extracts. Gel filtration analysis revealed a native molecular m ass of similar to 160 kDa for this binding activity. Our results point to a similar architecture between ciliate telomerase RNA and metazoan U6 small nuclear RNA promoters.