![]() On the other hand, even slight deficiencies of normal human telomerase activation during early development lead to disease by premature exhaustion of telomere-limited proliferation. Remarkably, ∼90% of human cancers have reactivated telomerase ( 4, 5). ![]() ![]() This repression of telomerase contributes to tumor suppression by limiting the proliferative capacity of any individual cell lineage. Although telomerase is active in every cell division of single-celled organisms, multicellular animals including humans largely silence telomerase after early embryogenesis, with complete repression or only tightly regulated transient expression in human somatic cells ( 2, 3). Telomerase is a ribonucleoprotein (RNP) reverse transcriptase specialized for restoring the telomeric repeats eroded from chromosome ends during genome replication ( 1). Our findings delineate roles for human telomerase RNA template-flanking regions, establish a biologically relevant pseudoknot-alternative RNA conformation, and expand the repertoire of human telomerase repeat synthesis. In cells, over-stabilization or destabilization of the alternative state severely inhibited biogenesis of active telomerase. Using mutations to reduce over-stability of the alternative conformation, we restore high activity and processivity to otherwise inactive altered-template telomerase ribonucleoproteins. We discovered that some altered-template sequences stabilize an alternative RNA conformation that precludes the pseudoknot by base-pairing of one pseudoknot strand to the template 3′ end. In comparison, requirements for the template sequence itself are confounding: different substitutions of the same position have strikingly different consequences, from improved processivity and activity to complete inactivation. Using human telomerase, we show that the length of template 3′-flanking single-stranded RNA is a determinant of repeat addition processivity whereas template 5′-flanking single-stranded RNA and P2a.1 are critical for activity but not processivity. Telomerase RNAs have single-stranded regions that separate the template from a 5′ stem and 3′ pseudoknot, and mammals gained additional stem P2a.1 separating the template from the pseudoknot. Telomerase adds telomeric repeats to chromosome ends by processive copying of a template within the telomerase RNA bound to telomerase reverse transcriptase.
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