The Molecular Mechanics of Eukaryotic Translation Initiation
- 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
- Antonio Munoz, BA, Predoctoral 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 made significant progress on several fronts this year. First, we completed our studies on the roles of the domains of the eukaryotic translation initiation factor eIF3 in promoting recruitment of mRNA and initiator tRNA to the ribosomal pre-translation initiation complex (PIC). Our data indicated that the mRNA entry channel arm of eIF3 as well as its b subunit play important roles in stabilizing binding of the initiator tRNA–containing eIF2 ternary complex to the PIC and in accelerating mRNA binding. The N-terminal domain of the a subunit of eIF3 plays an important role in stabilizing mRNA binding in the exit channel (Reference 1). These studies were done in collaboration with Alan Hinnebusch and Leoš Valášek.
In our studies of the role of the RNA–activated ATPase eIF4A in promoting mRNA recruitment to the PIC, we showed that the PIC itself activates the ATPase function of the factor over and above the stimulation provided by RNA. These data indicate that eIF4A interacts with the PIC. We also showed that eIF4A and its ATPase activity enhance loading of mRNAs onto the PIC regardless of their degree of secondary structure. In addition, structures in the 5′-untranslated region (UTR) and body of the mRNA contribute in a non-additive manner to inhibiting recruitment of mRNAs to the PIC and in imposing a requirement for eIF4A. These data are in contrast to prevailing models for mRNA recruitment, which suggest that eIF4A’s sole role is to unwind secondary structures in the 5′-UTRs of mRNAs. Our data indicate that eIF4A plays a key role in loading all mRNAs—even those with no expected secondary structure—and that it helps deal with the global structure of an mRNA rather than just secondary structures in the 5′-UTR. We are currently preparing a manuscript describing these studies.
We are also nearing completion of our studies of the effect of temperature on start codon recognition in yeast. We used a combination of reporter-based assays and genome-wide ribosome profiling to show that temperature affects initiation at start codons upstream of the canonical AUG in many mRNAs, but that the direction of the change is different for different messages and correlates with the length of and degree of structure in the 5′-UTR. A manuscript describing this work is planned. These studies are complemented by ribosome profiling experiments determining the genome-wide effects of mutations in translation initiation factors that increase or decrease the fidelity of start codon recognition.
In addition to these projects, we are making progress by studying the mechanism of action of the DEAD box RNA helicase Ded1p. Our work indicates that Ded1p operates in a distinct manner that depends on the mRNA being loaded onto the PIC. In collaboration with Venki Ramakrishnan's and Alan Hinnebusch’s groups, we have also continued our studies on the mechanism of action of the factor eIF5 in start codon recognition. We have also started a series of high-throughput experiments to determine the mRNA features that control the rate of translation initiation in yeast.
Additional Funding
- Funding is provided via a Memorandum of Understanding (MOU) between NIGMS and NICHD.
Publications
- Aitken CE, Beznosková P, Vlckova V, Chiu W-L, Zhou F, Valášek LS, Hinnebusch AG, Lorsch JR. Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex. eLife 2016;5:e20934.
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
- Leoš S. Valášek, PhD, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic
Contact
For more information, email jon.lorsch@nih.gov.
