Translating experience: thinking outside the box.

Tony Hunter is Director of the Salk Institute Cancer Center, 10010 N Torrey Pines Road, San Diego, California 92037, USA. e-mail: hunter@salk.edu acetyl group. Had we been primed to think outside the box, we might have discovered this initiation mechanism sooner. In 1970, Richard and I were using rabbit reticulocytes to analyse the fate of nascent globin chains released prematurely from ribosomes following puromycin treatment. We found that these chains were rapidly degraded; this also occurred in vitro when isolated ribosomes incubated with puromycin were added to a reticulocyte lysate supplemented with ATP. Just before I left for my postdoctoral position at the Salk Institute, we presented these results at a 1971 meeting and, in a proceedings chapter, speculated that proteases unable to recognize intact folded globin chains degraded these truncated proteins. Had we continued the project and thought of alternative explanations, we might have uncovered the ATP-dependent protease activity, reported by Fred Goldberg in 1977 and later shown to be due to the proteasome, and the ubiquitin-mediated protein degradation system, reported by Avram Hershko and Aaron Ciechanover in 1978 — both using rabbit reticulocyte lysates. In 1973, when I returned to the Department of Biochemistry in Cambridge following my postdoc, a new willingness to think laterally led to an unexpected discovery. When a fire destroyed our lab in June 1974, we became refugees and were graciously offered temporary space in the New Addenbrookes site, opposite the MRC Laboratory of Molecular Biology (LMB). Our plight led Max Perutz, the Director of LMB, to offer us dining rights in the LMB cafeteria, where we ate lunch every day. The unspoken rule of the cafeteria was that you had to sit with people from other groups, and this soon led to Tim Hunt and myself collaborating with John Knowland and David Zimmern in the LMB tobacco mosaic virus (TMV) group, led by Aaron Klug. At the time, the packaged TMV genomic RNA was known to encode the 18 kDa viral coat protein, but all attempts to translate coat protein in vitro had failed, generating instead much larger 140–160 kDa products. We found the same when we translated TMV virion RNA in the wheat germ system we were using to study protein synthesis initiation, or in Xenopus laevis oocytes. Eventually, we realized that as TMV-infected tobacco plants make large amounts of coat protein, their leaves must contain a coat protein mRNA. One of the first experiments we did in our new home was to translate total RNA isolated from TMV-infected tobacco leaves using wheat germ samples stored in a liquid N2 freezer that had survived the fire. Gratifyingly, we obtained authentic coat protein, and demonstrated that its mRNA corresponded to the 750 bases at the 3' end of the genomic RNA. We had discovered that TMV expresses its coat protein through a subgenomic viral RNA, a strategy commonly adopted by RNA viruses for expressing individual proteins from polycistronic RNAs. Our findings did not explain why fragments from the 3' end of the genomic RNA failed to translate coat protein. This mystery was solved in 1975, when a 5' methylated cap structure was shown to be required for eukaryotic mRNA translation. This initiation mechanism restricts translation to the cistron adjacent to the cap, explaining why internal cistrons, such as the coat protein gene, cannot be translated. We now know that the virion RNA has a cap, as does the subgenomic RNA made in infected cells. These early experiences stood me in good stead when I fortuitously stumbled across a novel phospho-amino acid generated in an in vitro kinase assay using polyoma virus middle T antigen. This was not the expected phospho-serine or phospho-threonine, and only by being willing to ignore dogma did I surmise that this might be phospho-tyrosine. The rest, as they say, is history.