The Ribosome: A Structural Biology Triumph Offering New Horizons (2019)

The symposium and memorial service at Yale, January 18–19, 2019, informed all in attendance, either fully knowing or in need of being reminded, of the extraordinary scientific talent of Thomas Steitz (Fig. 1), as well as his skill and devotion as a mentor, the affection of his peers, and his consistent radiation of upbeat joy to friends, close colleagues, and family. Several engaging memoirs of Tom Steitz were published soon after his untimely passing, and because of the greater space allowed for one (1), it can be recommended as the most comprehensive for those who wish to read a very complete picture of Tom's accomplishments and persona. Using ribosomes from the Dead Sea archaebacterium Haloarcula marismortui, first crystallized by Ada Yonath and colleagues, Steitz and his group pushed ahead with great persistence and innovativeness. He, Venki Ramakrishnan, and Yonath shared the 2009 Nobel Prize in Chemistry. Ribosomes were first recognized in the mid-1950s by several groups that fractionated cells and sought the sites of protein synthesis, including Arthur Pardee and Howard Schachman at the University of California, Berkeley, Paul Zamecnik at Harvard Medical School, Alexander Spirin in Moscow, and George Palade and Phil Siekevitz at Rockefeller University. Given their content of RNA, comprising about half their weight, these cytoplasmic particles were named “ribosomes” by Richard Roberts at the Carnegie Institution of Washington, also among the first to work on them. For readers wishing a comprehensive summary of research on ribosomes, an excellent account is that by H.-J. Rhineberger (2). Once it was recognized that ribosomes were the machines of protein synthesis and thus executed completion of the pathway that Francis Crick dubbed the Central Dogma (DNA makes DNA, DNA makes RNA, RNA makes protein), interest promptly arose as to their structure as the logical pathway to a detailed understanding of their function. The essence of this, it was later discovered, was both to engage a site in mRNA (3) and then to ferry aminoacylated transfer RNAs through three distinct sites, at one of which the catalytic step of a peptide bond occurs. As Harry Noller said at the symposium, for him the most beguiling aspect of the ribosome is this translocation process, coupled to the chemical step. It is also worth noting here that to Steitz's credit and astute sense of what problems are important, he and his group worked not only on the ribosome but also solved the structures of enzymes involved in DNA replication, transcription, and the aminoacylation-tRNA coupling step of protein synthesis, thus contributing in major ways to the structural biology of every step in the Central Dogma. Prior to the X-ray crystallographic advances, there were of course key chapters in understanding ribosome structure. One, pioneered by Masayasu Nomura at the University of Wisconsin, was to try to take ribosomes apart and reconstitute them onto the RNA with separated proteins. At the time, this was thought by some to be unproductive because it was imagined that the ribosome's function was an emergent property of non-commutative components that had been assembled in some mysterious way, never to be undone without scrambling everything up in attempted test tube reassembly. But Nomura succeeded, and this was a huge step both conceptually and heuristically. In hindsight, I think the skepticism was warranted because it had to do with a belief that the proteins of the ribosome were so interwoven, both with one another and with the RNA with all its folds, as to be a tangle that would resist dissociation without destruction of either the proteins or the RNA. There were also questions about whether the RNA or the proteins were on the surface or each buried together within. The atomic structures later revealed that most of the protein is on the outside. To paraphrase Peter Moore of the Yale group, speaking on National Public Radio's All Things Considered the week the 50S sub unit structure was published: The proteins have their globular heads on the surface with fingers descending into a great cavern of RNA. I am reminded here that Herman Wouk in his novel Inside, Outside (4) wrote of a sweater given to his main character, David Goodkind, that had a dangling thread, so inviting. After succumbing to the temptation, all that was left was a tangle of wool. Not so for the ribosome. At this time, several investigators were making antibodies to ribosomal proteins, the most successful of whom was Georg Stöffler in the large group headed by Heinz-Günter Wittmann in Berlin. A major step was that of James Lake at the University of California, Los Angeles, who used antibodies to tag ribosomes and achieved the first immune-electron microscopy images of certain proteins in or on the ribosome. These were of course nowhere near the atomic resolution that was later to come, and yet the overall shape they conveyed was correct and thus turned out to be very helpful as the crystallographic maps started to arrive. Soon thereafter, Peter Moore and Donald Engelman at Yale used neutron scattering to obtain more detailed structural information, and this too was a key step in the precrystallographic era of ribosome structural biology. Attempts to crystallize ribosomes were underway at this time, including ones by Alexander Spirin in Moscow and the aforementioned Wittmann group in Berlin. It was Ada Yonath, working with both the Wittmann group and her own lab at the Weizmann Institute, who got diffracting crystals from the ribosomes of a Dead Sea archaebacterium, but her subsequent efforts to get high-resolution diffraction maps progressed slowly. Further details of how she, Tom Steitz's group, and those of Venki Ramakrishnan at the University of Cambridge and Harry Noller at the University of California, Santa Cruz, moved successfully ahead are beyond the scope of this piece and can be pursued by interested readers in Ramakrishnan's engaging account (5). For our present purposes, it is sufficient to say that the structures of the Steitz and Ramakrishnan groups revealed unanticipated details of the chemistry of protein synthesis, as did the structure and experimental work of Noller's group to understand the tRNA translocation mechanism in atomic detail. A seminal insight from these combined contributions is that the ribosome's active site, called the peptidyl transferase center, involves RNA-based chemistry, i.e., the ribosome is a ribozyme. Among many other thoughts that came to me during the symposium and memorial, too numerous and some too personally emotional to relate here, was that Tom would have been the last, given his intrepid thirst for new knowledge, to say that the ribosome story is finished. At the time of his death, stirring new developments were taking place. One is the increasing evidence for ribosome heterogeneity and its functional significance (6). Another is the ingenious design of “orthogonal ribosomes” in which directed evolution of the two RNA components leads to synthesis of novel biopolymers (7). These are things Tom will sadly not see as they develop, and as yet other ribosome surprises are likely to arise. But of one thing we can be sure: he would love them as new dimensions and would tackle them mightily as to their structural biology roots. And he would, as always, come by asking his talented students and postdocs, “Have you solved your structure yet?” as so many of his former lab members recalled him doing in their remembrances at the symposium. What was clear from their recollections was that this question was not idle curiosity but intense interest for the team to succeed and a conveyance of both his encouragement and his passion.