Alan G. Hinnebusch

Alan G. Hinnebusch, a Distinguished Investigator at the National Institutes of Health, was interested in science from an early age. “I was always fascinated with being able to figure out how things work,” Hinnebusch says. “I’ve always had an affinity for biology.” 

During college at the University of Dayton, Hinnebusch took genetics, which sparked his interest in the subject. “I was fascinated by the fact that you could follow the inheritance of fairly simple traits and learn an awful lot about why individuals vary in a species. As I got into molecular genetics, I started reading about the pioneering work that had been done in bacteria on gene regulation,” Hinnebusch says. 

The instructor for genetics, Ken McDougall, ended up becoming a mentor for Hinnebusch. “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,” Hinnebusch says. 

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 in Lynn Klotz’s laboratory. “It didn’t make a lot of sense that it would have huge amounts of repeated DNA involved in regulating gene expression and development, because they’re unicellular and very ancient,” Hinnebusch says. 

During his time in graduate school, cloning was invented, which meant scientists could now study a single gene in great detail. Hinnebusch, who recognized how transformational this was, hurried up to finish his Ph.D., with a goal of studying gene regulation in more advanced eukaryotes.

For his postdoctoral training, he chose to study with Gerald R. Fink, who had developed a number of tools to study gene regulation in budding yeast. “Yeast had already been developed as a really powerful genetic system,” Hinnebusch says. “Classical genetics existed, and then after transformation was invented by Fink, you could do molecular genetics. You could go in and clone a gene, if you have a mutation in it.”

In Fink’s lab, 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 and clone GCN4. 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.” 

During his follow-up experiments, Hinnebusch noticed something unusual about the GCN4 gene. “Upstream of the gene, there was all this extra sequence that wasn’t encoding anything for the protein, but with the unusual feature of having 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 additional AUGs, and how would they interact with one another?” Hinnebusch says. 

In a series of follow-up experiments, Hinnebusch and his post-doctoral fellow Peter Mueller were able to construct mutations in each of the AUGs. They discovered that each AUG formed a small upstream Open Reading Frame, or uORF for short, which acted as either a positive or negative element that determined whether or not GCN4 was translated. “We 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 translation in amino acid-starved cells.

Eventually, Hinnebusch and his fellow Jean-Pierre Abastado discovered that these uORFs control the translation of GCN4 by dictating whether or not the ribosome stays on the messenger RNA after translating the uORFs. “We now call this the delayed reinitiating mechanism,” Hinnebusch says. 

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 and his fellows Thomas Dever and Ronald Wek 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 they 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 protein 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 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 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 the biosynthetic genes.

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

This pathway was later named the Integrated Stress Response by David Ron, and has been found to be broadly conserved, from yeast to humans. It has also been shown to play an important role in a number of processes, and is a target for potential drug therapies. 

“What's fascinating to me now is that many others have discovered that this stress response is extremely important in many different biological settings, including in humans,” Hinnebusch says. This includes being linked to certain diseases, as well as being implicated in memory consolidation.