Active yeast ribosome preparation using monolithic anion exchange chromatography

Munoz, Antonio; Yourik, Paul; Rajagopal, Vaishnavi; Nanda, Jagpreet S.; Lorsch, Jon R.; Walker, Sarah E.

doi:10.1080/15476286.2016.1270004

Cited by 19 publications

(13 citation statements)

References 21 publications

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“…As previously described, eIF3 was expressed in yeast [8], but here a Nitrogen mill is employed for highly reproducible lysis [20]. Figure 1 shows the improved eIF3 purification scheme with representative chromatograms and gels.…”

Section: Resultsmentioning

confidence: 99%

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

Liu

Schuessler

Sahoo

et al. 2019

Methods

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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.

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Section: Resultsmentioning

confidence: 99%

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

Liu

Schuessler

Sahoo

et al. 2019

Methods

Self Cite

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“…mRNAs were capped (m 7 GpppG) in vitro using vaccinia virus D1/D12 capping enzyme with either α- 32 P radiolabeled GTP (Perkin Elmer) or unlabeled GTP (for pulse-chase) ( Mitchell et al, 2010 ). Eukaryotic initiation factors, 40S ribosomal subunits, and WT Ded1 and its mutant derivatives were expressed and purified as described previously ( Acker et al, 2007 ; Mitchell et al, 2010 ; Munoz et al, 2017 ; Gupta et al, 2018 ).…”

Section: Methodsmentioning

confidence: 99%

Distinct interactions of eIF4A and eIF4E with RNA helicase Ded1 stimulate translation in vivo

Gulay

Gupta

Lorsch

et al. 2020

eLife

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Yeast DEAD-box helicase Ded1 stimulates translation initiation, particularly of mRNAs with structured 5'UTRs. Interactions of the Ded1 N-terminal domain (NTD) with eIF4A, and Ded1-CTD with eIF4G, subunits of eIF4F, enhance Ded1 unwinding activity and stimulation of preinitiation complex (PIC) assembly in vitro. However, the importance of these interactions, and of Ded1-eIF4E association, in vivo were poorly understood. We identified separate amino acid clusters in the Ded1-NTD required for binding to eIF4A or eIF4E in vitro. Disrupting each cluster selectively impairs native Ded1 association with eIF4A or eIF4E, and reduces cell growth, polysome assembly, and translation of reporter mRNAs with structured 5'UTRs. It also impairs Ded1 stimulation of PIC assembly on a structured mRNA in vitro. Ablating Ded1 interactions with eIF4A/eIF4E unveiled a requirement for the Ded1-CTD for robust initiation. Thus, Ded1 function in vivo is stimulated by independent interactions of its NTD with eIF4E and eIF4A, and its CTD with eIF4G.

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“…Eukaryotic initiation factors- eIF1, eIF1A, eIF2, eIF3, eIF4A, eIF4B, eIF4G·4E (WT and mutants), eIF5- were expressed and purified as described previously ( Acker et al, 2007 ; Mitchell et al, 2010 ; Rajagopal et al, 2012 ). 40S ribosomal subunits were prepared as described in ( Munoz et al, 2017 ). Recombinant Ded1 proteins (N-terminal His 6 -tag, pET22b vector)- WT Ded1(1-604), Ded1 E307A , Ded1-

CTD (1-535) and Ded1-

NTD (117-604) were purified as described previously ( Gao et al, 2016 ; Hilliker et al, 2011 ) with some modifications.…”

Section: Methodsmentioning

confidence: 99%

Yeast Ded1 promotes 48S translation pre-initiation complex assembly in an mRNA-specific and eIF4F-dependent manner

Gupta

Lorsch

Hinnebusch

2018

eLife

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DEAD-box RNA helicase Ded1 is thought to resolve secondary structures in mRNA 5'-untranslated regions (5'-UTRs) that impede 48S preinitiation complex (PIC) formation at the initiation codon. We reconstituted Ded1 acceleration of 48S PIC assembly on native mRNAs in a pure system, and recapitulated increased Ded1-dependence of mRNAs that are Ded1-hyperdependent in vivo. Stem-loop (SL) structures in 5'-UTRs of native and synthetic mRNAs increased the Ded1 requirement to overcome their intrinsically low rates of 48S PIC recruitment. Ded1 acceleration of 48S assembly was greater in the presence of eIF4F, and domains mediating one or more Ded1 interactions with eIF4G or helicase eIF4A were required for efficient recruitment of all mRNAs; however, the relative importance of particular Ded1 and eIF4G domains were distinct for each mRNA. Our results account for the Ded1 hyper-dependence of mRNAs with structure-prone 5'-UTRs, and implicate an eIF4E·eIF4G·eIF4A·Ded1 complex in accelerating 48S PIC assembly on native mRNAs.

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Active yeast ribosome preparation using monolithic anion exchange chromatography

Cited by 19 publications

References 21 publications

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

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

Distinct interactions of eIF4A and eIF4E with RNA helicase Ded1 stimulate translation in vivo

Yeast Ded1 promotes 48S translation pre-initiation complex assembly in an mRNA-specific and eIF4F-dependent manner

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