Using mechanical unfolding by optical tweezers (OT) and steered molecular dynamics (SMD) simulations, we have demonstrated the critical role of Mg 2+ ions for the resistance of the Beet Western Yellow Virus (BWYV) pseudoknot (PK) to unfolding. The two techniques were found to be complementary, providing information at different levels of molecular scale. Findings from the OT experiments indicated a critical role of stem 1 for unfolding of the PK, which was confirmed in the SMD simulations. The unfolding pathways of wild type and mutant appeared to depend upon pH and nucleotide sequence. SMD simulations support the notion that the stability of stem 1 is critical for -1 frameshifting. The all-atom scale nature of the SMD enabled clarification of the precise role of two Mg2+ ions, Mg45 and Mg52, as identified in the BWYV X-ray crystallography structure, in -1 frameshifting. On the basis of simulations with "partially" and "fully" hydrated Mg 2+ ions, two possible mechanisms of stabilizing stem 1 are proposed. In both these cases Mg2+ ions play a critical role in stabilizing stem 1, either by directly forming a salt bridge between the strands of stem 1 or by stabilizing parallel orientation of the strands in stem 1, respectively. These findings explain the unexpected drop in frameshifting efficiency to null levels of the C8U mutant in a manner consistent with experimental observations.
ASJC Scopus subject areas
- Colloid and Surface Chemistry