Specific length and structure rather than high thermodynamic stability enable regulatory mRNA stem-loops to pause translation

Chen Bao,Hironao Wakabayashi,David H Mathews,Dmitri N Ermolenko,Inna Nykonchuk,Mingyi Zhu

doi:10.1038/s41467-022-28600-5

Abstract

Translating ribosomes unwind mRNA secondary structures by three basepairs each elongation cycle. Despite the ribosome helicase, certain mRNA stem-loops stimulate programmed ribosomal frameshift by inhibiting translation elongation. Here, using mutagenesis, biochemical and single-molecule experiments, we examine whether high stability of three basepairs, which are unwound by the translating ribosome, is critical for inducing ribosome pauses. We find that encountering frameshift-inducing mRNA stem-loops from the E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) hinders A-site tRNA binding and slows down ribosome translocation by 15-20 folds. By contrast, unwinding of first three basepairs adjacent to the mRNA entry channel slows down the translating ribosome by only 2-3 folds. Rather than high thermodynamic stability, specific length and structure enable regulatory mRNA stem-loops to stall translation by forming inhibitory interactions with the ribosome. Our data provide the basis for rationalizing transcriptome-wide studies of translation and searching for novel regulatory mRNA stem-loops.

Highlights

Translating ribosomes unwind mRNA secondary structures by three basepairs each elongation cycle
Another study suggested that the effects of mRNA secondary structure on translational efficiency measured by ribosome profiling may be masked by the presence of codons that are decoded by abundant tRNAs in the structured regions of ORFs17
We used a combination of single-molecule Förster resonance energy transfer (smFRET) and biochemical assays to investigate properties of frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) that are important for inducing ribosome pausing and −1 programmed ribosomal frameshifting (PRF)

Summary

Introduction

Translating ribosomes unwind mRNA secondary structures by three basepairs each elongation cycle. Optical tweezer experiments suggested that ribosome translocation through three consecutive GC basepairs is only 2–3-fold slower than translocation along unpaired RNA10,12 Consistent with these data, several transcriptome-wide ribosome profiling analyses performed in yeast and E. coli cells indicated that most of the secondary structure elements within coding regions of mRNA do not produce detectable ribosome pauses or significantly change translational efficiency[14,15]. In contrast to aforementioned reports demonstrating modest impact of mRNA secondary structure on translation rate, a ribosome profiling study performed in E. coli revealed strong negative correlation between translational efficiency and the presence of extensive intramolecular basepairing interactions in ORFs suggesting that both initiation and elongation phases of protein synthesis can be regulated by mRNA secondary structure[16]. Certain mRNA stem-loops and pseudoknots are known to trigger programmed translational pauses[20] and stimulate −1 programmed ribosomal frameshifting (PRF) by slowing down the ribosome21. −1 PRF plays an important role in regulating synthesis of bacterial, viral and eukaryotic proteins, including translation of gag-pol polyprotein of human immunodeficiency virus (HIV)[22] and C-terminally extended polyprotein in SARS-CoV-2 coronavirus, which caused the COVID-19 pandemic[23,24]

Methods

Results

Conclusion