MRNA stem-loops can pause the ribosome by hindering A-site tRNA binding.

Chen Bao,Andrei A Korostelev,Sarah Loerch,Clarence Ling,Nikolaus Grigorieff,Dmitri N Ermolenko

doi:10.7554/elife.55799

Abstract

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

Highlights

During translation elongation, the ribosome moves along mRNA in a codon-by-codon manner while the mRNA is threaded through the mRNA channel of the small ribosomal subunit
The γ subunit is produced by a -1 programmed ribosome frameshifting (PRF) event that occurs with 50-80% efficiency
Upstream of the SD sequence, the dnaX_Slip mRNA contains a 25 nucleotide-long sequence complementary to a biotin-derivatized DNA oligonucleotide used to tether the mRNA to a microscope slide for single-molecule Förster resonance energy transfer (smFRET) experiments (Fig. 1)

Summary

Introduction

The ribosome moves along mRNA in a codon-by-codon manner while the mRNA is threaded through the mRNA channel of the small ribosomal subunit. The translating ribosome must unfold mRNA secondary structure to feed single-stranded mRNA through the narrow mRNA channel. The elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to pause or stall ribosome movement along mRNA. The α subunit of the signal recognition particle receptor is cotranslationally targeted to the endoplasmic reticulum membrane by a mechanism that requires a translational pause induced by an mRNA stem-loop structure (Young and Andrews, 1996). Ribosome pausing induced by mRNA structures accompanies -1 programmed ribosome frameshifting (PRF), which controls expression of a number of proteins in bacteria, viruses and eukaryotes (Caliskan et al, 2015). -1 PRF regulates synthesis of DNA polymerase III in bacteria (Tsuchihashi and Kornberg, 1990); gag-pol proteins of Human Immunodeficiency Virus (HIV) (Jacks et al, 1988), and HIV cytokine receptor ccr in higher eukaryotes (Belew et al, 2014)

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