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.
Eukaryotic translation initiation is a critical node at which gene expression is regulated. Among the factors that are known to regulate this process, DEAD‐box RNA helicases are believed to promote translation by resolving secondary structure in the 5′ UTR of an mRNA. These helicases include the protein Ded1 in budding yeast, which is required to remove cap‐distal structure in the 5′ UTR of an mRNA, thus enabling scanning of the preinitiation complex. Yet the role of Ded1 in translation is complex; a translationally repressive role has also been demonstrated by Ded1 overexpression. Further confounding investigations of Ded1 function are the large number of protein‐protein and protein‐RNA contacts made by Ded1 to regulate activity. To study the diverse roles that Ded1 plays in translation, we have developed a system that reports on Ded1‐specific changes in translation in vivo. This system relies on a fluorescence‐based reporter that simultaneously promotes the transcription of two mRNA transcripts: one encoding RFP with a 5′ UTR that is translated independent of Ded1 activity, and the other encoding GFP with a 5′ UTR that requires Ded1‐activity for translation. We are using this system in combination with a library of random Ded1 mutants to broadly interrogate Ded1 activities for effects on translation initiation and repression.
Support or Funding Information
This work was supported by NIH grant R00GM119173 and start‐up funds from SUNY at Buffalo, College of Arts and Sciences.
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