Crowding Promotes the Switch from Hairpin to Pseudoknot Conformation in Human Telomerase RNA

TL;DR

This study demonstrates through molecular simulations that molecular crowding stabilizes the pseudoknot conformation of human telomerase RNA, potentially restoring activity in mutants with destabilized pseudoknots.

Contribution

It introduces a coarse-grained simulation model to show how crowding influences RNA conformational equilibrium, highlighting effects of crowder size and mixture composition.

Findings

Crowding enhances pseudoknot stability over hairpin in telomerase RNA.

Small crowders increase stability more than larger ones.

Crowder mixtures show additive effects on melting temperature.

Abstract

Formation of a pseudoknot in the conserved RNA core domain in the ribonucleoprotein human telomerase is required for function. In vitro experiments show that the pseudoknot (PK) is in equilibrium with an extended hairpin (HP) structure. We use molecular simulations of a coarse-grained model, which reproduces most of the salient features of the experimental melting profiles of PK and HP, to show that crowding enhances the stability of PK relative to HP in the wild type and in a mutant associated with dyskeratosis congenita. In monodisperse suspensions, small crowding particles increase the stability of compact structures to a greater extent than larger crowders. If the sizes of crowders in a binary mixture are smaller than the unfolded RNA, the increase in melting temperature due to the two components is additive. In a ternary mixture of crowders that are larger than the unfolded RNA, which mimics the composition of ribosome, large enzyme complexes and proteins in E. coli, the marginal increase in stability is entirely determined by the smallest component. We predict that crowding can restore partially telomerase activity in mutants, which dramatically decrease the PK stability.