The eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome.

Initiation of translation is the process by which initiator tRNA and the start codon of mRNA are positioned in the ribosomal P site. In eukaryotes, one of the first steps involves the binding of two small factors, eIF1 and eIF1A, to the small (40S) ribosomal subunit. This facilitates tRNA binding, allows scanning of mRNA, and maintains fidelity of start codon recognition. Using cryo-EM, we have obtained 3D reconstructions of 40S bound to both eIF1 and eIF1A, and with each factor alone. These structures reveal that together, eIF1 and eIF1A stabilize a conformational change that opens the mRNA binding channel. Biochemical data reveal that both factors accelerate the rate of ternary complex (eIF2*GTP*Met-tRNA(i)(Met)) binding to 40S but only eIF1A stabilizes this interaction. Our results suggest that eIF1 and eIF1A promote an open, scanning-competent preinitiation complex that closes upon start codon recognition and eIF1 release to stabilize ternary complex binding and clamp down on mRNA.

N- and C-terminal residues of eIF1A have opposing effects on the fidelity of start codon selection.

Translation initiation factor eIF1A stimulates preinitiation complex (PIC) assembly and scanning, but the molecular mechanisms of its functions are not understood. We show that the F131A,F133A mutation in the C-terminal tail (CTT) of eIF1A impairs recruitment of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex to 40S subunits, eliminating functional coupling with eIF1. Mutating residues 17-21 in the N-terminal tail (NTT) of eIF1A also reduces PIC assembly, but in a manner rescued by eIF1. Interestingly, the 131,133 CTT mutation enhances initiation at UUG codons (Sui(-) phenotype) and decreases leaky scanning at AUG, while the NTT mutation 17-21 suppresses the Sui(-) phenotypes of eIF5 and eIF2beta mutations and increases leaky scanning. These findings and the opposite effects of the mutations on eIF1A binding to reconstituted PICs suggest that the NTT mutations promote an open, scanning-conducive conformation of the PIC, whereas the CTT mutations 131,133 have the reverse effect. We conclude that tight binding of eIF1A to the PIC is an important determinant of AUG selection and is modulated in opposite directions by residues in the NTT and CTT of eIF1A.

The eIF1A C-terminal domain promotes initiation complex assembly, scanning and AUG selection in vivo.

Translation initiation factor 1A stimulates 40S-binding of the eukaryotic initiation factor 2 (eIF2)/GTP/Met-tRNA(iMet) ternary complex (TC) and promotes scanning in vitro. eIF1A contains an OB-fold present in bacterial IF1 plus N- and C-terminal extensions. Truncating the C-terminus (deltaC) or mutating OB-fold residues (66-70) of eIF1A reduced general translation in vivo but increased GCN4 translation (Gcd- phenotype) in a manner suppressed by overexpressing TC. Consistent with this, both mutations diminished 40S-bound TC, eIF5 and eIF3 in vivo, and deltaC impaired TC recruitment in vitro. The assembly defects of the OB-fold mutation can be attributed to reduced 40S-binding of eIF1A, whereas deltaC impairs eIF1A function on the ribosome. A substitution in the C-terminal helix (98-101) also reduced 43S assembly in vivo. Rather than producing a Gcd- phenotype, however, 98-101 impairs GCN4 derepression in a manner consistent with defective scanning by reinitiating ribosomes. Indeed, 98-101 allows formation of aberrant 48S complexes in vitro and increases utilization of non-AUG codons in vivo. Thus, the OB-fold is crucial for ribosome-binding and the C-terminal domain of eIF1A has eukaryotic-specific functions in TC recruitment and scanning.

Inhibition of protein synthesis by aminoglycoside-arginine conjugates.

Inhibition of translation by small molecule ligands has proven to be a useful tool for understanding this complex cellular mechanism, as well as providing drugs of significant medical importance. Many small molecule ligands inhibit translation by binding to RNA or RNA/protein components of the ribosomal subunits and usurping their function. A class of peptidomimetics [aminoglycoside-arginine conjugates (AAC)] has recently been designed to inhibit HIV TAR/tat interaction and in experiments aimed at assessing the inhibitory effects of AACs on TAR-containing transcripts, we found that AACs are general inhibitors of translation. Experiments reported herein aim at characterizing these novel properties of AACs. We find that AACs are inhibitors of eukaryotic and prokaryotic translation and exert their effects by blocking peptide chain elongation. Structure/activity relationship studies suggest that inhibition of translation by AACs is directly related to the number of arginine groups present on the aminoglycoside backbone and to the nature of the core aminoglycoside. AACs are therefore attractive tools for understanding and probing ribosome function.