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

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 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.

ArfB can displace mRNA to rescue stalled ribosomes

“…Rotation of the head relative to the body (also known as "head swivel") can achieve~20°. The opening between these 30S domains allows mRNA to sample conformations outside the mRNA tunnel 34,35 . ArfB dimerization likely shifts the mRNA dynamic equilibrium by fixing longer mRNAs outside the mRNA tunnel.…”

“…This indicates that mRNA downstream the P-site codon is conformationally unstable in the absence of a stabilizing A-site interaction. Furthermore, downstream mRNA with high propensity for secondary-structure formation can depart from the mRNA tunnel and fold in the A site 34,40 . It is possible therefore, that in addition to the truncated and rare-codon substrates, ArfB also recognizes no-go complexes with structured mRNA downstream the tunnel.…”

RNA-responsive elements for eukaryotic translational control

“…6). Similar fold changes upon induction while retaining the use of the T7 RNAP and promoter could be achieved by using a bicistronic design and adding stop codons and stem loops 23 before the IRES modules (Extended Data Fig. 7a).…”

“…Therefore, we decided to explore methods of reducing translational activity in the presence of canonical 5′ capping. By adding stop codons and stem loops 23 after a gene controlled by a constitutive promoter, we were able to reduce the basal expression of downstream mRNA despite the reliance on RNA polymerase II (Fig. 2c,d).…”

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

“…The slippery sequence allows cognate or near-cognate pairing of the P-site and A-site tRNAs in both 0 and −1 frames and thus makes frameshift thermodynamically favorable 26 . A number of previous studies demonstrated that when the slippery sequence is bound in the P and A sites of the ribosome and the FSS is positioned at the entry to mRNA tunnel of the small subunit, the rate of tRNA/mRNA translocation decreases by approximately an order of magnitude 11,[27][28][29][30][31] . We also recently demonstrated that, when positioned 11-12 nucleotides downstream of the P-site codon, FSSs can transiently escape the ribosome helicase and inhibit A-site tRNA binding by docking into the A site of the small (30S) ribosomal subunit 31 .…”