Two inactive fragments of the integral RNA cooperate to assemble active telomerase with the human protein catalytic subunit (hTERT) in vitro

We have mapped the 5' and 3' boundaries of the region of the human telomera se RNA (hTR) that is required to produce activity with the human protein ca talytic subunit (hTERT) by using in vitro assembly systems derived from rab bit reticulocyte lysates and human cell extracts. The region spanning nucle otides +33 to +325 of the 451-base hTR is the minimal sequence required to produce levels of telomerase activity that are comparable with that made wi th full-length hTR. Our results suggest that the sequence approximately 270 bases downstream of the template is required for efficient assembly of act ive telomerase in vitro; this sequence encompasses a substantially larger p ortion of the 3' end of hTR than previously thought necessary. In addition, we identified two fragments of hTR (nucleotides +33 to +147 and +164 to +3 25) that cannot produce telomerase activity when combined separately with h TERT but can function together to assemble active telomerase. These results suggest that the minimal sequence of hTR can be divided into two sections, both of which are required for de novo assembly of active telomerase in vi tro.