Discrimination against RNA Backbones by a ssDNA Binding Protein

Abstract

Pot1 is the shelterin component responsible for the protection of the single-stranded DNA (ssDNA) overhang at telomeres in nearly all eukaryotic organisms. The C-terminal domain of the DNA-binding domain, Pot1pC, exhibits non-specific ssDNA recognition, achieved through thermodynamically equivalent alternative binding conformations. Given this flexibility, it is unclear how specificity for ssDNA over RNA, an activity required for biological function, is achieved. Examination of the ribose-position specificity of Pot1pC shows that ssDNA specificity is additive but not uniformly distributed across the ligand. High-resolution structures of several Pot1pC complexes with RNA-DNA chimeric ligands reveal Pot1pC discriminates against RNA by utilizing non-compensatory binding modes that feature significant rearrangement of the binding interface. These alternative conformations, accessed through both ligand and protein flexibility, recover much, but not all, of the binding energy, leading to the observed reduction in affinities. These findings suggest that intermolecular interfaces are remarkably sophisticated in their tuning of specificity toward flexible ligands.

Single-stranded telomere-binding protein employs a dual rheostat for binding affinity and specificity that drives function

“… 35 ), which have suggested that the nucleic acid-binding interface is malleable. For example, single-stranded recognition interfaces can be remodeled to match different substrates to achieve specific recognition for both DNA and RNA, as exemplified by the Oxytricha nova telomere end-binding protein ( 36 ), the S. pombe Pot1 protein ( 28 , 37 ), and, in RNA recognition, the PUF protein ( 38 ) and the MS2 coat protein ( 39 ). This malleability could be dynamic in origin ( 40 , 41 ).…”