Tetrahymena telomerase protein p65 induces conformational changes throughout telomerase RNA (TER) and rescues telomerase reverse transcriptase and TER assembly mutants.
The biogenesis of the Tetrahymena telomerase ribonucleoprotein particle (RNP) is enhanced by p65, a La family protein. Single-molecule and biochemical studies have uncovered a hierarchical assembly of the RNP, wherein the binding of p65 to stems I and IV of telomerase RNA (TER) causes a conformational change that facilitates the subsequent binding of telomerase reverse transcriptase (TERT) to TER. We used purified p65 and variants of TERT and TER to investigate the conformational rearrangements that occur during RNP assembly. Nuclease protection assays and mutational analysis revealed that p65 interacts with and stimulates conformational changes in regions of TER beyond stem IV. Several TER mutants exhibited telomerase activity only in the presence of p65, revealing the importance of p65 in promoting the correct RNP assembly pathway. In addition, p65 rescued TERT assembly mutants but not TERT activity mutants. Taken together, these results suggest that p65 stimulates telomerase assembly and activity in two ways. First, by sequestering stems I and IV, p65 limits the ensemble of structural conformations of TER, thereby presenting TERT with the active conformation of TER. Second, p65 acts as a molecular buttress within the assembled RNP, mutually stabilizing TER and TERT in catalytically active conformations.
Berman
Andrea J
AJ
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Gooding
Anne R
AR
Cech
Thomas R
TR
D001483:Base Sequence; D017125:Genes, Protozoan; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019098:Telomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016808:Tetrahymena thermophila / Q000235:genetics* / Q000378:metabolism*
D009687:Nuclear Proteins; C101880:Pdd1 protein, Tetrahymena; D010750:Phosphoproteins; D015800:Protozoan Proteins; D016053:RNA, Protozoan; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase