SPECIFIC RNA RESIDUE INTERACTIONS REQUIRED FOR ENZYMATIC FUNCTIONS OFTETRAHYMENATELOMERASE

The ribonucleoprotein enzyme telomerase is a specialized reverse trans criptase that synthesizes telomeric DNA by copying a template sequence within the telomerase RNA. Here we analyze the actions of telomerase from Tetrahymena thermophila assembled in vivo with mutated or wild-ty pe telomerase RNA to define further the roles of particular telomerase RNA residues involved in essential enzymatic functions: templating, s ubstrate alignment, and promotion of polymerization. Position 49 of th e telomerase RNA defined the 3' templating residue boundary, demonstra ting that seven positions, residues 43 to 49, are capable of acting as templating residues. We demonstrate directly that positioning of the primer substrate involves Watson-Crick base pairing between the primer with telomerase RNA residues. Unexpectedly, formation of a Watson-Cri ck base pair specifically between the primer DNA and telomerase RNA re sidue 50 is critical in promoting primer elongation. In contrast, muta nt telomerase with the cytosine at position 49 mutated to a G exhibite d efficient 3' mispair extension. This work provides new evidence for specific primer-telomerase interactions, as well as base-specific inte ractions involving the telomerase RNA, playing roles in essential acti ve-site functions of telomerase.