2026 Gruber Genetics Prize

Alan G. Hinnebusch, a Distinguished Investigator at the National Institutes of Health, first became interested in genetics during his time as an undergraduate at the University of Dayton. “My undergraduate genetics professor, Ken McDougall, was very influential,” Hinnebusch says. “His course was probably the hardest course you could take as a biology major, but he also exposed us to all of the latest things that were going on in molecular genetics.” 

With McDougall’s support, Hinnebusch decided to pursue a career in genetics, which included applying to graduate school. At McDougall’s suggestion, Hinnebusch applied to some of the bigger universities. “He told me to shoot high, and so I did, thinking I would never get in,” Hinnebusch says. Hinnebusch was then accepted to Harvard University for graduate school, where he got his Ph.D. in biochemistry and molecular biology. 

During graduate school, Hinnebusch studied repeated DNA in a primitive eukaryote. “It didn’t make a lot of sense that it would have huge amounts of repeated DNA, that were involved in regulating gene expression and development, because they’re so simple and very ancient,” Hinnebusch says. During his time as a graduate student, cloning was invented, which meant that it was now possible to study a specific gene in great detail. Recognizing how transformational this discovery was, Hinnebusch hurried to finish his PhD, so that he could direct his attention to studying gene regulation in higher order eukaryotes. 

After graduating with his Ph.D., Hinnebusch did his postdoctoral training in the lab of Gerald R. Fink, at Cornell University and Massachusetts Institute of Technology, where the development of DNA transformation had made budding yeast a powerful model for molecular genetics of eukaryotic cell biology. Hinnebusch studied the regulation of histidine biosynthesis, which is an amino acid that is essential for a number of processes. Fink had already isolated mutants defective for regulating histidine synthesis, which had resulted in the discovery that these genes were responsible for controlling the synthesis of other amino acids as well. “It was a global sort of cross-pathway regulation, rather than being specific for a single amino acid,” Hinnebusch says. “I thought that was very fascinating.”

During his time in Fink’s lab, Hinnebusch studied the genetic pathway responsible for regulating amino acid biosynthesis, by constructing double mutants combining mutations in positive- and negative-acting factors. “I realized that this classical approach of combining mutations would allow you to arrange genes in a pathway,” Hinnebusch says. “It’s called epistasis.” 

By the end of his postdoc, Hinnebusch had been able to order the positive- and negative-acting genes governing this general amino acid control into a linear pathway. With Fink’s blessing, Hinnebusch was able to take these mutations to his next position, as a scientist at the NIH, to continue the work he had done during his postdoc. “That’s a huge gift for your postdoctoral mentor to give,” Hinnebusch says. “Gerry was incredibly generous.”

The genes Hinnebusch was studying included GCN2 and GCN4, both positive regulators of amino acid biosynthesis, with GCN2 acting upstream of GCN4, and GCN4 functioning closest to the amino acid biosynthetic genes that get turned on in starved cells. During his follow-up experiments, Hinnebusch noticed something unusual about GCN4. “Upstream of the gene, there was all this extra sequence that wasn’t encoding anything for the protein, but it had the unusual feature of having these four additional AUG start codons, in a 600 nucleotide region, which was pretty much unheard of at that point,” Hinnebusch says. 

Intrigued by this finding, Hinnebusch decided to focus on answering the questions of what these extra AUGs were doing, and how they related to the translational control of GCN4. “The puzzle was, why would you need four of these AUGs, and how would they interact with one another?” Hinnebusch says. 

In a series of follow-up experiments, Hinnebusch was able to construct mutations in each of the AUGs. What he discovered was that each AUG formed a small upstream Open Reading Frame, or uORF for short, which acted either as a positive or negative element that determined whether or not GCN4 was translated. “I found that some of them acted as negative elements and some acted as positive elements,” Hinnebusch says. By analyzing uORF double mutants, epistasis revealed that uORF1 acted positively by overcoming the negative effects of uORFs 3 and 4, to stimulate GCN4 synthesis in amino acid-starved cells. 

Eventually, what Hinnebusch discovered was that these uORFs affect the translation of GCN4, by dictating whether or not the ribosome stays on the messenger RNA strand after translating the uORFs. “If, after you translate the first uORF, and the ribosome stays on the message, instead of falling off, it can reinitiate downstream. If it recovers the ability to reinitiate quickly, it will be forced to translate one of the inhibitory uORFs 3 or 4, and fall off the message. But if it takes longer to recover a factor needed for reinitiation, it may be able to skip over the inhibitory uORFs, keep scanning, and then pick up the required factor before reaching the GCN4 start codon,” Hinnebusch says. “We now call this the delayed reinitiation mechanism.”

Under normal conditions, when cells are not starved for amino acids, ribosomes translate the first uORF, after which they reinitiate quickly and get stuck at one of the other inhibitory uORFs. In starved cells, when amino acids are reduced in quantity, the time required to become competent for reinitiation increases, allowing a fraction of ribosomes to skip over the inhibitory uORFs and use the GCN4 start codon instead, synthesizing GCN4 protein. 

In later experiments, Hinnebusch looked at the role of eIF2, which acts as a translation initiation factor, in GCN4 translation. It was known that phosphorylation of the α-subunit of eIF2 lowers its ability to deliver initiator tRNA to the ribosome. As he eventually found, this provided a molecular switch that can be used in amino acid-starved cells to suppress global protein synthesis, while selectively activating translation of GCN4, through the uORFs. As GCN4 had been shown to induce the transcription of amino acid biosynthetic genes, this switch enables cells to produce more amino acids while conserving their existing supplies. 

“In starvation conditions, the kinase called GCN2 would be activated and phosphorylate eIF2, which would decrease the ability of eIF2 to deliver initiator tRNA to the ribosome. It would now take longer for these scanning ribosomes, after they translated uORF1, to regain the initiator tRNA needed to recognize the next AUG codon,” Hinnebusch says. This allows a fraction of ribosomes to skip over uORFs 3 and 4 but recover initiator tRNA in time to recognize the GCN4 start codon and produce the GCN4 protein needed to activate transcription of biosynthetic genes.

“You’re using what in other settings is a big stick to shut off protein synthesis, but using it here to tweak the system, to get a stress response,” Hinnebusch says.