Engineering cis-telomerase RNAs that add telomeric repeats to themselves.
Telomerase is a ribonucleoprotein complex consisting of a protein reverse transcriptase (TERT) and an RNA subunit (TR). Telomerase normally adds telomeric DNA repeats to chromosome ends. Here, we engineer human and Tetrahymena cis-telomerase RNAs, each having a DNA primer covalently linked to its 3' end. We find that cis-telomerase synthesizes DNA with increased repeat addition processivity (RAP) but does not completely rescue the RAP defect of the L14A mutant of Tetrahymena TERT. This supports the conclusion that L14 has a function beyond binding the DNA primer and preventing dissociation during multiple rounds of repeat addition. By comparing cis-telomerases with various linker lengths, we find that a 5 nt linker gives near-optimal activity, indicating that the distance between the 3' end of the telomerase RNA pseudoknot region and the 5' end of the DNA primer is approximately 33 A. Even a 2 nt linker (approximately 14 A) gives some activity, indicating a high degree of conformational flexibility in this ribonucleoprotein complex. More generally, the cis system will allow structure-function relationships of each RNA molecule to be read directly through the reaction that it performs on itself.
Qiao
Feng
F
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, Campus Box 215, University of Colorado, Boulder, CO 80309-0215, USA.
Goodrich
Karen J
KJ
Cech
Thomas R
TR
D000818:Animals; D005818:Genetic Engineering; D006801:Humans; D009154:Mutation / Q000235:genetics; D012313:RNA / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism*; D013768:Tetrahymena / Q000201:enzymology
C034477:RNA primers; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase