Sequence-specific and conformation-dependent binding of yeast telomerase RNA to single-stranded telomeric DNA.
Telomerase is a ribonucleoprotein reverse trans-criptase responsible for the maintenance of one strand of telomere terminal repeats. The mechanisms whereby telomerase recognizes chromosomal ends are not fully characterized. Earlier studies showed that the yeast telomerase RNP could bind the dG-rich strand of yeast telomeres with high affinity and sequence specificity. Further analysis of telomerase-telomere complex formation in vitro as described in this report led to the following conclusions. First, telomerase binding to short DNAs is magnesium-dependent, while binding to long DNAs is magnesium-independent, consistent with the existence of more than one interaction site. Second, binding is likely to be mediated largely through the RNA subunit of telomerase (TLC1), because de-proteinated TLC1 RNA also binds telomeres with high affinity and sequence specificity, and exhibits the same length and divalent cation dependence as telomerase RNP. The crucial role of RNA in binding is further supported by the ability of TLC1 transcripts synthesized in vitro to form stable complexes with telomeric DNA. Finally, results from deletion analysis and RNase H-mediated cleavage suggest that a specific conformation(s) of the RNA is required for stable binding, and that non-template regions of the TLC1 RNA may contribute directly or indirectly to the stability of the RNA-DNA complex.
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Department of Microbiology, W. R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA. nflue@mail.med.cornell.edu
D002413:Cations, Divalent; D017931:DNA Primers / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D019098:Telomerase / Q000737:chemistry / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism*; D014158:Transcription, Genetic
D002413:Cations, Divalent; D017931:DNA Primers; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D019098:Telomerase