Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation

Gabriel Demo,Egor Svidritskiy,Howard B Gamper,Ya-Ming Hou,Andrei A Korostelev,Isao Masuda,Christine E Carbone,Anna B Loveland

doi:10.1038/s41467-021-24911-1

Abstract

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.

Highlights

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses
+1FS controls the expression of the essential release factor 2 in bacteria[5,6], regulates metabolitedependent enzyme expression[7], and leads to pathological expression of huntingtin[8] in eukaryotes. +1FS can be amplified by dysregulation of ribosome quality control mechanisms[9,10], and it is being exploited to synthetically expand the coding repertoire of genomes by inserting non-natural amino acids via a tRNA that can perform +1FS11
While we previously showed that E. coli tRNAPro(UGG) performs +1FS in vitro[17], it remained unknown whether the tRNA performs +1FS in cells, where all isoacceptors are present, and whether its post-transcriptional modifications impact +1FS

Summary

Introduction

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. We performed cryo-EM analyses of ribosomes with nonframeshifting CCA-A or frameshifting CCC-A motifs, and first focused on the non-rotated pre-translocation structures (Fig. 3; “Methods”).

Results

Conclusion