Alternative Mode of E-Site tRNA Binding in the Presence of a Downstream mRNA Stem Loop at the Entrance Channel

Abstract

Structured mRNAs positioned downstream of the ribosomal decoding center alter gene expression by slowing protein synthesis. Here, we solved the cryo-EM structure of the bacterial ribosome bound to an mRNA containing a 3' stem loop that regulates translation. Unexpectedly, the E-site tRNA adopts two distinct orientations. In the first structure, normal interactions with the 50S and 30S E site are observed. However, in the second structure, although the E-site tRNA makes normal interactions with the 50S E site, its anticodon stem loop moves ∼54 Å away from the 30S E site to interact with the 30S head domain and 50S uL5. This position of the E-site tRNA causes the uL1 stalk to adopt a more open conformation that likely represents an intermediate state during E-site tRNA dissociation. These results suggest that structured mRNAs at the entrance channel restrict 30S subunit movement required during translation to slow E-site tRNA dissociation.

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

“…1). In this ribosome complex, the second Lys codon of the slippery sequence is positioned in the A site, and the FSS is expected to be one nucleotide downstream of the entrance to the mRNA channel (Yusupova et al, 2001;Zhang et al, 2018). Consistent with previous reports, ribosomes containing P-site peptidyl-tRNA (N-Ac-Val-Lys-tRNA Lys ) are predominately in the NR (0.6 FRET) state ( Fig.…”

A tandem active site model for the ribosomal helicase

“…The now‐unpaired nucleotides of the 5′ strand of the stem would become immobilized by binding to protein S3, while those of the 3′‐strand of the stem would become flexible. Consistent with this prediction, chemical probing of an mRNA hairpin placed at the tunnel entrance revealed low backbone flexibility in the first 3–4 nt of the 5′ strand, but high backbone flexibility in the complementary 3′‐strand nucleotides .…”

“…The now-unpaired nucleotides of the 5 0 strand of the stem would become immobilized by binding to protein S3, while those of the 3 0 -strand of the stem would become flexible. Consistent with this prediction, chemical probing of an mRNA hairpin placed at the tunnel entrance revealed low backbone flexibility in the first 3-4 nt of the 5 0 strand, but high backbone flexibility in the complementary 3 0 -strand nucleotides [13]. According to this model, a stable structure can hinder translocation in two ways: in the distal segment, it can delay reverse head rotation until either unwinding or stick-slip occurs; in the proximal segment, if sufficiently stable, it can delay forward head rotation until unwinding occurs (Fig.…”

“…Based on clues from structural observations (Fig. ) , the proposed model for the ribosomal helicase envisions two active sites operating in tandem to unwind encountered mRNA structures (Figs and ) and forms the basis for a kinetic scheme for translocation over these structures (Fig. ).…”

“…Recent structural studies have revealed the binding of three nucleotides of single‐stranded mRNA to ribosomal protein S3 just outside of the tunnel . The backbone of the bound mRNA is extended and straightened, deviating significantly from the curved A‐form geometry (Fig.…”