Conserved domains within the RNA component of telomerase provide the template for reverse transcription, recruit protein components to the holoenzyme and are required for enzymatic activity. Among the functionally essential domains in ciliate telomerase RNA is stem-loop IV, which strongly stimulates telomerase activity and processivity even when provided in trans. The NMR structure of Tetrahymena thermophila stem-loop IV shows a highly structured distal stem-loop linked to a conformationally flexible template-proximal region by a bulge that severely kinks the entire RNA. Through extensive structure-function studies, we identify residues that contribute to both these structural features and to enzymatic activity, with no apparent effect on the binding of TERT protein. We propose that the bending induced by the GA bulge and the flexibility of the template-proximal region allow positioning of the prestructured apical loop during the catalytic cycle.

The telomerase ribonucleoprotein complex ensures complete replication of eukaryotic chromosomes. Telomerase RNA, TER, provides the template for replicating the G-rich strand of telomeric DNA, provides an anchor site for telomerase-associated proteins, and participates in catalysis through several incompletely characterized mechanisms. A major impediment towards understanding its non-templating roles is the absence of high content structural information for TER within the telomerase complex. Here, we used selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) to examine the structure of Tetrahymena TER free in solution and bound to tTERT in the minimal telomerase RNP. We discovered a striking difference in the two conformations and established direct evidence for base pair triples in the tTER pseudoknot. We then used SHAPE data, previously published FRET data, and biochemical inference to model the structure of tTER using discrete molecular dynamics simulations. The resulting tTER structure was docked with a homology model of tTERT to characterize the conformational changes of tTER that attend binding to tTERT. Free in solution, tTER appears to contain four pairing regions: stems I, II, and IV, which are present in the commonly accepted structure, and stem III, a large paired region that encompasses the template and pseudoknot domains. Our interpretation of the data and subsequent modeling affords a molecular model for telomerase assemblage in which a large stem III of tTER unwinds to allow proper association of the template with the tTERT active site and formation of the pseudoknot. Additionally, analysis of our SHAPE data and previous enzymatic footpinting allows us to propose a model for stem-loop IV function in which tTERT is activated by binding stem IV in the major grove of the helix-capping loop.

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Telomerase is a ribonucleoprotein complex that reverse transcribes a portion of its RNA subunit during the synthesis of G-rich DNA at the 3' end of each chromosome in most eukaryotes. This activity compensates for the inability of the normal DNA replication machinery to fully replicate chromosome termini. The roles of telomerase in cellular immortality and tumor biology have catalyzed a significant interest in this unusual polymerase. Recently the first structures of two domains, the CR4/CR5 and pseudoknot, of human telomerase RNA (hTR) were reported, offering a structural basis for interpreting biochemical studies and possible roles of hTR mutations in human diseases. Structures of the stem II and stem IV domains of Tetrahymena thermophila TR as well as the N-terminal domain of the T. thermophila telomerase reverse transcriptase have also been determined. These studies complement previous biochemical studies, providing rich insight into the structural basis for telomerase activity.

Telomerase is a specialized reverse transcriptase, which catalyzes the addition of telomeric repeats to the 3' ends of linear chromosomes using its integral RNA subunit as the template. An active Tetrahymena thermophila telomerase complex can be reconstituted in vitro from two essential components, tTERT, the catalytic protein subunit, and tTR, the RNA subunit. While the sequence specificity of telomerase has been investigated using template sequence mutants, there is no information regarding its backbone specificity. To address this question, we engineered two mutant forms of the telomerase RNA subunit that contain DNA only in the templating region and used rabbit reticulocyte lysates to reconstitute telomerase activity with the chimeric tTRs. The resultant telomerase mutants were able to extend telomeric DNA primers, albeit with reduced efficiency compared to the wild type. The reduced activity is presumed to be a function of the nascent DNA-template duplex structure. Additionally, the DNA-dependent telomerase mutants were RNase-sensitive, confirming that nontemplate portions of tTR are critical for maintaining activity of the telomerase ribonucleoprotein complex even after it is assembled. The splint ligation approach that we outline will allow the generation of tTR mutants containing a variety of nucleotide analogues, facilitating more elaborate studies of the interactions between the telomerase template and active site.