The Molecular Mechanics of Eukaryotic Translation Initiation
- Jon Lorsch, PhD, Chief, Laboratory on the Mechanism and Regulation of Protein Synthesis
- Fujun Zhou, PhD, Research Fellow
- Meizhen Hou, MS, Biologist
- Julie Bocetti, BA, Postbaccalaureate Intramural Research Training Award Fellow
The goal of our research 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 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 preinitiation 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, events that 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, which is conducive to scanning, to a closed one, which 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.
We continued our studies of the mechanism of translation initiation using our recently developed Rec-Seq transcriptome–wide approach. Rec-Seq utilizes our fully reconstituted yeast translation initiation system and is able to simultaneously monitor the recruitment of each mRNA in the yeast transcriptome to the 43S ribosomal PIC. We spent considerable effort this year honing the method to provide reproducible, quantitative results. A central goal was the development of a reliable approach to normalize the sequence read data to an absolute standard, so that direct comparisons can be made across experiments and replicates. After trying a number of different strategies, we believe we have finally found a way to perform this normalization step using a “spike in,” consisting of PICs assembled on luciferase mRNAs, which is added in a standard concentration to each experimental sample before processing. We also repeated our previous experiments to examine the function of the RNA helicase Ded1, using additional replicates to test reproducibility. This replication experiment yielded results similar to those of the original experiments and confirmed that Ded1 preferentially enhances mRNA recruitment to the 43S PIC for mRNAs with long, structured 5′-untranslated regions. We also performed an experiment to assess the effects of varying 40S ribosomal subunit and mRNA concentrations relative to each other. Initial results appeared to support a previously proposed model that limiting 40S subunit concentrations would specifically disfavor translation of “weak” mRNAs that compete poorly for binding to the PIC compared with “strong,” more competitive mRNAs. However, we will reanalyze these data using our new normalization approaches to determine how this affects the results and conclusions.
Figure 1. Dependency of mRNA recruitment on Ded1
Click image to view.
Ded1 specifically enhances recruitment of mRNAs with long 5′-untranslated regions (5′-UTRs).
Additional Funding
- Funding is provided via a Memorandum of Understanding (MOU) between NIGMS and NICHD.
Publication
- Gulay S, Gupta N, Lorsch JR, Hinnebusch AG. Distinct interactions of eIF4A and eIF4E with RNA helicase Ded1 stimulate translation in vivo. eLife 2020;9:e58243.
Collaborators
- Thomas Dever, PhD, Section on Protein Biosynthesis, NICHD, Bethesda, MD
- Alan Hinnebusch, PhD, Section on Nutrient Control of Gene Expression, NICHD, Bethesda, MD
- Venkatraman Ramakrishnan, PhD, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
Contact
For more information, email jon.lorsch@nih.gov or visit https://irp.nih.gov/pi/jon-lorsch.
