SummaryDuring eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2α, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

Background: Start codon recognition triggers eIF1 and P i release from the preinitiation complex. Results: The C-terminal tail of eIF1A moves closer to eIF5 upon start codon recognition, and this movement is required for P i release. Conclusion: eIF1 release and movement of the eIF1A C-terminal tail toward eIF5 are coupled processes. Significance: Start codon recognition induces coordinated movements of initiation factors that trigger downstream events.

The maturation of RNAs includes site-specific post-transcriptional modifications that contribute significantly to hydrogen bond formation within RNA and between different RNAs, especially in formation of mismatch base pairs. Thus, an understanding of the geometry and strength of the base-pairing of modified ribonucleoside 59-monophosphates, previously not defined, is applicable to investigations of RNA structure and function and of the design of novel RNAs. The geometry and free energies of base-pairings were calculated in aqueous solution under neutral conditions with AMBER force fields and molecular dynamics simulations (MDSs). For example, unmodified uridines were observed to bind to uridine and cytidine with significant stability, but the ribose C19-C19 distances were far short (;8.9 Å ) of distances observed for canonical A-form RNA helices. In contrast, 5-oxyacetic acid uridine, known to bind adenosine, wobble to guanosine, and form mismatch base pairs with uridine and cytidine, bound adenosine and guanosine with geometries and energies comparable to an unmodified uridine. However, the 5-oxyacetic acid uridine base paired to uridine and cytidine with a C19-C19 distance comparable to that of an A-form helix, ;11 Å , when a H 2 O molecule migrated between and stably hydrogen bonded to both bases. Even in formation of canonical base pairs, intermediate structures with a second energy minimum consisted of transient H 2 O molecules forming hydrogen bonded bridges between the two bases. Thus, MDS is predictive of the effects of modifications, H 2 O molecule intervention in the formation of base-pair geometry, and energies that are important for native RNA structure and function.

Eukaryotic initiator tRNA (tRNA i ) contains several highly conserved unique sequence features, but their importance in accurate start codon selection was unknown. Here we show that conserved bases throughout tRNA i , from the anticodon stem to acceptor stem, play key roles in ensuring the fidelity of start codon recognition in yeast cells. (PICs). Disrupting the C3:G70 base pair in the acceptor stem produces a Sui -phenotype and also reduces the rate of TC binding to 40S subunits in vitro and in vivo. Both defects are suppressed by an Ssu -substitution in eIF1A that stabilizes the open/P OUT conformation of the PIC that exists prior to start codon recognition. Our data indicate that these signature sequences of tRNA i regulate accuracy by distinct mechanisms, promoting the open/P OUT conformation of the PIC (for C3:G70) or destabilizing the closed/P IN state (for G31:C39 and A54) that is critical for start codon recognition.

In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes.

Eukaryotic translation initiation factor (eIF) 5 acts as a GTPase activating protein for eIF2 that carries the first tRNA onto the ribosome. Recent data have shown that eIF5 antagonizes eIF1, enhancing its release from the PIC in response to start codon recognition. eIF1 release promotes the conversion of the PIC from open conformation that scan the mRNA to closed one arrested on the start codon. Recent data indicate that an interaction occurs between eIF1A and eIF5 upon start codon recognition by PIC. We observed fluorescence energy transfer (FRET) between fluorescently labeled derivatives of eIF1A and eIF5 within the PIC that is strongly enhanced upon start codon recognition. Our data indicate that the C‐terminal tail (CTT) of eIF1A moves closer to the N‐terminal region of eIF5 upon start codon recognition. This conformational change is involved in stabilizing the closed state of PIC. The kinetics of this FRET change mirror the kinetics of eIF1 release from the PIC on start codon recognition, suggesting two events are coupled. Mutations in eIF1A CTT that decrease the fidelity of start codon recognition in vivo uncouple eIF1 release from eIF1A CTT movement towards eIF5 NTD as well as from release of inorganic phosphate from eIF2. Our results suggest a model in which eIF1 release from PIC promotes movement of the eIF1A CTT towards eIF5 NTD that in turn promotes Pi release from eIF2, converting it to its inactive, GDP‐bound state.