Ciliate telomerase RNA loop IV nucleotides promote hierarchical RNP assembly and holoenzyme stability.
Telomerase adds simple-sequence repeats to chromosome 3' ends to compensate for the loss of repeats with each round of genome replication. To accomplish this de novo DNA synthesis, telomerase uses a template within its integral RNA component. In addition to providing the template, the telomerase RNA subunit (TER) also harbors nontemplate motifs that contribute to the specialized telomerase catalytic cycle of reiterative repeat synthesis. Most nontemplate TER motifs function through linkage with the template, but in ciliate and vertebrate telomerases, a stem-loop motif binds telomerase reverse transcriptase (TERT) and reconstitutes full activity of the minimal recombinant TERT+TER RNP, even when physically separated from the template. Here, we resolve the functional requirements for this motif of ciliate TER in physiological RNP context using the Tetrahymena thermophila p65-TER-TERT core RNP reconstituted in vitro and the holoenzyme reconstituted in vivo. Contrary to expectation based on assays of the minimal recombinant RNP, we find that none of a panel of individual loop IV nucleotide substitutions impacts the profile of telomerase product synthesis when reconstituted as physiological core RNP or holoenzyme RNP. However, loop IV nucleotide substitutions do variably reduce assembly of TERT with the p65-TER complex in vitro and reduce the accumulation and stability of telomerase RNP in endogenous holoenzyme context. Our results point to a unifying model of a conformational activation role for this TER motif in the telomerase RNP enzyme.
Robart
Aaron R
AR
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720-3200, USA.
O'Connor
Catherine M
CM
Collins
Kathleen
K
D004795:Enzyme Stability; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016053:RNA, Protozoan / Q000737:chemistry / Q000378:metabolism*; D019098:Telomerase / Q000737:chemistry / Q000378:metabolism*; D016808:Tetrahymena thermophila / Q000201:enzymology / Q000378:metabolism*
D016053:RNA, Protozoan; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase