A stem-loop of Tetrahymena telomerase RNA distant from the template potentiates RNA folding and telomerase activity.
The ribonucleoprotein enzyme telomerase adds telomeric repeats to the ends of linear chromosomes. The Tetrahymena telomerase reverse transcriptase (TERT) protein and the telomerase RNA can be reconstituted into an active complex in vitro in rabbit reticulocyte lysates. We have probed the structure of the telomerase RNA in the reconstituted complex with RNases T1 and V1. Upon TERT binding to the RNA, sites of both protection and enhancement of cleavage were observed, suggesting potential protein-binding sites and conformational changes in the RNA. Especially prominent was a large region of RNase V1 protection in stem-loop IV. A number of loop IV mutants still bound TERT but showed drastic decreases in the level of telomerase activity and the loss of protein-dependent folding of the pseudoknot region of the telomerase RNA. The telomerase activity defect and the misfolding of the pseudoknot were partially separable, leading to the proposal of two functions for stem-loop IV: to aid in the folding of the pseudoknot and to function more directly in the active site of telomerase. Thus an RNA element far from the template makes a major contribution to Tetrahymena telomerase enzyme activity.
Sperger
J M
JM
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Cech
T R
TR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D017384:Sequence Deletion; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D016808:Tetrahymena thermophila / Q000201:enzymology* / Q000235:genetics; D013942:Thymine Nucleotides / Q000378:metabolism
D004268:DNA-Binding Proteins; D003848:Deoxyguanine Nucleotides; C034477:RNA primers; D016053:RNA, Protozoan; D013942:Thymine Nucleotides; D012313:RNA; C029603:deoxyguanosine triphosphate; D019098:Telomerase; D004722:Endoribonucleases; C032825:ribonuclease V(1); C024157:thymidine 5'-triphosphate