Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for -1 ribosomal frameshifting stimulation.

Zhensheng Zhong,J Jeya Vandana,Haiping Zhang,Jiahao Shi,Lixia Yang,Do Thuy Uyen Ha Lam,Gang Chen,Lanyuan Lu,René C L Olsthoorn

doi:10.1038/srep39549

Zhensheng Zhong, J Jeya Vandana + Show 7 more

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https://doi.org/10.1038/srep39549

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Abstract

Minus-one ribosomal frameshifting is a translational recoding mechanism widely utilized by many RNA viruses to generate accurate ratios of structural and catalytic proteins. An RNA pseudoknot structure located in the overlapping region of the gag and pro genes of Simian Retrovirus type 1 (SRV-1) stimulates frameshifting. However, the experimental characterization of SRV-1 pseudoknot (un)folding dynamics and the effect of the base triple formation is lacking. Here, we report the results of our single-molecule nanomanipulation using optical tweezers and theoretical simulation by steered molecular dynamics. Our results directly reveal that the energetic coupling between loop 2 and stem 1 via minor-groove base triple formation enhances the mechanical stability. The terminal base pair in stem 1 (directly in contact with a translating ribosome at the slippery site) also affects the mechanical stability of the pseudoknot. The −1 frameshifting efficiency is positively correlated with the cooperative one-step unfolding force and inversely correlated with the one-step mechanical unfolding rate at zero force. A significantly improved correlation was observed between −1 frameshifting efficiency and unfolding rate at forces of 15–35 pN, consistent with the fact that the ribosome is a force-generating molecular motor with helicase activity. No correlation was observed between thermal stability and −1 frameshifting efficiency.

Highlights

Recent advances in the discovery and understanding of complex RNA structures, dynamics, and functions have dramatically changed our view of RNA’s role in biology
An mRNA structure located downstream of the slippery sequence separated by a single-stranded spacer (2 to 8 nucleotides in length) stimulates −1 frameshifting from 0 frame X XXY YYZ to −1 frame XXX YYY Z
Chemical mapping (Footprinting) studies[29] of beet western yellow virus (BWYV) pseudoknot-E coli ribosome complexes show that the first base pair in stem 1 of a pseudoknot may melt in the presence of a ribosome at the slippery site if the single-stranded spacer is shortened from 6 to 5 nt

Summary

Introduction

Recent advances in the discovery and understanding of complex RNA structures, dynamics, and functions have dramatically changed our view of RNA’s role in biology. We focus on understanding how the translation reading frame can be regulated by cis-acting mRNA structures. The shifting of the mRNA reading frame of a megadalton ribosome complex can be manipulated by cis-acting mRNA sequences and structures. An mRNA structure located downstream of the slippery sequence separated by a single-stranded spacer (2 to 8 nucleotides (nt) in length) stimulates −1 frameshifting from 0 frame X XXY YYZ to −1 frame XXX YYY Z. In the process of unfolding of the downstream structure, the ribosome has a probability to slip into an alternative frame at the slippery site, eventually resulting in the accommodation of a new cognate aminoacyl-tRNA on the first codon in the −1 frame. Ritchie et al proposed a mechanism in which the frequency of the formation of alternative mRNA structures is proportional to −1 frameshifting efficiency[38]

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