Reconstitution and analyses of RNA interactions with eukaryotic translation initiation factors and ribosomal preinitiation complexes

Abstract

Control of translation initiation plays a critical role in the regulation of gene expression in all organisms, yet the mechanics of translation initiation in eukaryotic organisms are not well understood. Confounding studies of translation are the large number and overlapping functions of many initiation factors in cells, and a lack of cap-dependence in many in vitro systems. To shed light on intricate mechanisms that are often obscured in vivo, we use a fully reconstituted translation initiation system for analyzing RNA interactions with eukaryotic translation initiation factors and complexes from the model organism Saccharomyces cerevisiae. This system exhibits strong cap dependence, and dependence on translation factors varies with mRNA 5' UTR sequences as expected from genome-wide studies of translation. Here we provide optimized protocols for purification and analysis of labeled and unlabeled mRNA recruitment factors on both the rate and factor dependence of mRNA recruitment to the translation preinitiation complex in response to RNA sequence-and structure-changes. In addition to providing streamlined and detailed protocols, we provide a new construct for purification of higher yields of fluorescently labeled and unlabeled full-length eIF4G.

Deletion of the N-Terminal Domain of Yeast Eukaryotic Initiation Factor 4B Reprograms Translation and Reduces Growth in Urea

“…40S subunit (and 80S/polysome) containing fractions were verified from the same samples by blotting yeast ribosomal small subunit protein Rps2 (Aviva ARP63572_P050, 1:2000 dilution.) Rabbit antibodies to purified recombinant yeast eIF4A (1:20,000 dilution) and eIF4G1 (1:1000 dilution), generated by Invitrogen/Pierce custom antibody services and verified against the recombinant proteins, were used to determine the position of those proteins within gradients ( Liu et al, 2019 ). Each experiment was repeated three or more times from biological replicates.…”

Dynamic Phosphorylation Regulates Eukaryotic Translation Initiation Factor 4A Activity During the Cell Cycle

“…Polyclonal anti-eIF4A antibody was further purified from 40 ml of serum by running over a 5 ml Protein A column (Acrobiosystems Inc., MA-0422-C5) for immunoprecipitation experiments. Custom antibodies were verified against recombinant protein (23) and optimized for linear behavior using lysates from BY4741 yeast. Antibodies were used at the following concentrations for western blotting: rabbit anti-eIF4A (1:20000 dilution, custom), rabbit anti-eIF4G (1:1000, custom), and mouse anti-HA (1:5000, Invitrogen, 26183).…”

“…Polyclonal anti-eIF4A antibody was further purified from 40 ml of serum by running over a 5 ml Protein A column (Acrobiosystems Inc., MA-0422-C5) for immunoprecipitation experiments. Custom antibodies were verified against recombinant protein (23) and optimized for linear behavior using lysates from BY4741 yeast.…”

mRNA- and factor-driven dynamic variability controls eIF4F-cap recognition for translation initiation

“…The plasmid containing recombinant full-length eIF4G1 was a gift from Sarah Walker (also available on Addgene as plasmid #122248). This full-length eIF4G1 construct with a C-terminal chitin binding domain fusion was purified according to a published procedure (Liu et al, 2019), with the following modifications: the cells were lysed using a sonicator, and the final protein after elution was stored in 250 mM KCl instead of 250 mM KOAc, skipping the dialysis after the anionexchange chromatography step in the procedure, and DTT for storage was substituted with 2.5 mM TCEP. Briefly, E. coli cells expressing full-length eIF4G1 were thawed, and lysed using a sonicator after resuspending in Intein Lysis Buffer (50 mM HEPES-KOH, pH 7.4, 500 mM KCl, 1 mM EDTA).…”