National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2017 Annual Report of the Division of Intramural Research

The Molecular Mechanics of Eukaryotic Translation Initiation

Jon Lorsch
  • Jon Lorsch, PhD, Chief, Laboratory on the Mechanism and Regulation of Protein Synthesis
  • Jagpreet Nanda, PhD, Staff Scientist
  • Fujun Zhou, PhD, Research Fellow
  • Colin Aitken, PhD, Postdoctoral Intramural Research Training Award Fellow
  • Shardul Kulkarni, PhD, Postdoctoral Intramural Research Training Award Fellow
  • Daoming Qin, PhD, Postdoctoral Intramural Research Training Award Fellow
  • Paul Yourik, BA, Predoctoral Intramural Research Training Award Fellow
  • Charm Karunasiri, BA, Postbaccalaureate Student

The goal of our research group is to elucidate the molecular mechanisms underlying the initiation phase of protein synthesis in eukaryotic organisms. We use the yeast Saccharomyces cerevisiae as a model system and employ a range of approaches—from genetics to biochemistry to structural biology—in collaboration with Alan Hinnebusch's and Tom Dever’s labs here at NICHD and several other research groups around the world.

Eukaryotic translation initiation is a key control point in the regulation of gene expression. It begins when an initiator methionyl tRNA (Met-tRNAi) is loaded onto the small (40S) ribosomal subunit. Met-tRNAi binds to the 40S subunit as a ternary complex (TC) with the GTP–bound form of the initiation factor eIF2. Three other factors, eIF1, eIF1A, and eIF3, also bind to the 40S subunit and promote the loading of the TC. The resulting 43S pre-initiation complex (PIC) is then loaded onto the 5′-end of an mRNA with the aid of eIF3 and the eIF4 group of factors: the RNA helicase eIF4A; the 5′-7-methylguanosine cap-binding protein eIF4E; the scaffolding protein eIF4G; and the 40S subunit– and RNA–binding protein eIF4B. Both eIF4A and eIF4E bind to eIF4G and form the eIF4F complex. Once loaded onto the mRNA, the 43S PIC is thought to scan the mRNA in search of an AUG start codon. The process is ATP–dependent and likely requires several RNA helicases, including the DEAD–box protein Ded1p. Recognition of the start site begins with base pairing between the anticodon of tRNAi and the AUG codon. Base pairing then triggers downstream events that commit the PIC to continuing initiation from that point on the mRNA. These events include ejection of eIF1 from its binding site on the 40S subunit, movement of the C-terminal tail (CTT) of eIF1A, and release of phosphate from eIF2, which converts eIF2 to its GDP–bound state. In addition, the initiator tRNA moves from a position that is not fully engaged in the ribosomal P site [termed P(OUT)] to one that is [P(IN)], and the PIC as a whole converts from an open conformation that is conducive to scanning to a closed one that is not. At this stage, eIF2•GDP dissociates from the PIC, and eIF1A and a second GTPase factor, eIF5B, coordinate joining of the large ribosomal subunit to form the 80S initiation complex. In a process that appears to result in conformational reorganization of the complex, eIF5B hydrolyzes GTP and then dissociates along with eIF1A.

The molecular mechanics of eukaryotic translation initiation

We completed our studies of the mechanism of action of the DEAD-box ATPase eIF4A in promoting mRNA recruitment to the eukaryotic translation pre-initiation complex (PIC). Our work showed that eIF4A stimulates recruitment of mRNAs to the PIC regardless of their degree of secondary structure. The result indicates a role for the factor beyond unwinding structures in the 5′-untranslated regions (5′-UTRs) of mRNAs. We also showed that the PIC, and the eIF3i and eIF3j subunits of eIF3 in particular, stimulate the ATPase activity of eIF4A. In addition, our data showed that mRNA structure on the 3′-side of the start codon inhibits mRNA recruitment in a manner relieved by eIF4A’s ATPase activity, and that structure in the 5′-UTR and 3′ to the start codon synergistically inhibit mRNA recruitment. Taken together, the results suggest a model whereby eIF4A acts to disrupt the overall structure of an mRNA rather than specific, stable secondary structures in the 5′-UTR. In addition, our data suggest that eIF4A plays a role in loading all mRNAs into the entry channel of the PIC, perhaps by modulating the conformation of the 40S ribosomal subunit. A manuscript describing this work is currently under revision.

We are also working on a manuscript describing our genome-wide analysis of the effects of temperature on the utilization of the cognate and near-cognate start codons of upstream open reading frames (uORFs) in yeast. Our data show that several mRNAs have uORFs that are differentially translated depending on temperature, although there is no global change in fidelity of start codon recognition. Translation of some uORFs is activated at higher growth temperatures, while translation of others is inhibited. The same dichotomy also holds true at reduced growth temperatures. The organization of the uORF within the 5′-UTR appears to exert an influence on this temperature-dependent regulation of translation.

We completed a collaborative project with Alan Hinnebusch to study the role of the small ribosomal subunit protein Rps3/uS3. Our results indicated that two arginine residues in the protein make stabilizing contacts with mRNA in the entry channel of the 40S subunit, augmenting a similar role played by eIF3 (Reference 2).

We also completed a collaborative project with Alan Hinnebusch, Venki Ramakrishnan, and Adesh Saini to elucidate the structural and mechanistic basis for the role of the N-terminal domain of eIF5 in start codon recognition. A manuscript is currently in preparation describing this study.

We are beginning work on a manuscript about the mechanism of action of the DEAD-box RNA-dependent ATPase Ded1. The work shows that Ded1’s mechanism of action is distinct on different mRNAs and provides support for the proposal that the factor plays an important role in unwinding long, structured 5′-UTRs to promote mRNA loading onto the PIC.

Additional Funding

  • Funding is provided via a Memorandum of Understanding (MOU) between NIGMS and NICHD.

Publications

  1. Munoz AM, Yourik P, Rajagopal V, Nanda JS, Lorsch JR, Walker SE. Active yeast ribosome preparation using monolithic anion exchange chromatography. RNA Biol 2017 14:188-196.
  2. Dong J, Aitken CE, Thakur A, Shin BS, Lorsch JR, Hinnebusch AG. Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons. Proc Natl Acad Sci USA 2017 114:E2126-E2135.

Collaborators

  • Thomas Dever, PhD, Section on Protein Biosynthesis, NICHD, Bethesda, MD
  • Alan Hinnebusch, PhD, Section on Nutrient Control of Gene Expression, NICHD, Bethesda, MD
  • Nicholas Ingolia, PhD, University of California at Berkeley, Berkeley, CA
  • Venkatraman Ramakrishnan, PhD, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
  • Adesh Saini, PhD, Shoolini University, Solan, India
  • Leoš S. Valášek, PhD, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic

Contact

For more information, email jon.lorsch@nih.gov.

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