A novel RNA binding domain in tetrahymena telomerase p65 initiates hierarchical assembly of telomerase holoenzyme.
Telomerase reverse transcriptase (TERT) and telomerase RNA (TER) assemble as part of a holoenzyme that synthesizes telomeric repeats at chromosome ends. Genetic approaches have identified proteins that are required for in vivo association of TERT and TER, including the Tetrahymena telomerase holoenzyme protein p65. Here, we use quantitative assays to define the mechanisms underlying p65 function in holoenzyme biogenesis. We demonstrate that four modules of p65 contribute affinity for TER, including a C-terminal domain that recognizes the conserved dinucleotide bulge of central stem IV. This C-terminal domain is necessary and sufficient for p65's function in enhancing the recruitment of TERT to TER. Finally, we show that p65 and TERT assemble on TER with hierarchical rather than cooperative binding. These findings elucidate an extensive network of p65-TER recognition specificity and define a novel p65 RNA binding domain that initiates telomerase holoenyzme biogenesis.
O'Connor
Catherine M
CM
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3204, USA.
Collins
Kathleen
K
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D020035:Holoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012313:RNA / Q000378:metabolism; D016053:RNA, Protozoan / Q000378:metabolism*; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016808:Tetrahymena thermophila / Q000201:enzymology* / Q000235:genetics
D004268:DNA-Binding Proteins; D020035:Holoenzymes; D021122:Protein Subunits; D016053:RNA, Protozoan; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase