RNA recognition by the DNA end-binding Ku heterodimer.

RNA (New York, N.Y.) (2013-04-24)
Andrew B Dalby, Karen J Goodrich, Jennifer S Pfingsten, Thomas R Cech
ABSTRACT

Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem-loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem-loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dimethyl sulfate, ≥99.5%
Sigma-Aldrich
Dimethyl sulfate, ≥99%
Sigma-Aldrich
N-Cyclohexyl-N′-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate, 95%
Sigma-Aldrich
Dimethyl sulfate, puriss. p.a., ≥99.0% (GC)

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