December 2013

Harry F. Noller’s ‘Reflections’

One, two, three dimensions of ribosome function

Side by side photos of Harry F. Noller
Left: Harry Noller in his laboratory at UCSC as an assistant professor in 1970.
Right: Noller on sabbatical in Cambridge in April 1976, holding the autoradiogram of his first successful DNA sequencing gel.

Harry F. Noller reflects in The Journal of Biological Chemistry on his lifelong pursuit of cracking the functionality of the ribosome, via its structure, culminating in the aha moment when a “chubby L-shaped density appeared in exactly the position that we had predicted for the A site.”
Noller’s story starts more than half a century earlier, during his childhood in East Bay, Calif., in the era of the atomic bomb, the science fiction of Robert Heinlein and Arthur C. Clarke, and local newspaper headlines touting revelations on “the secret of life” in the test-tube reconstitution of the tobacco mosaic virus. Musings on these and other elements of science, both fantastic and real, marked Noller’s youth. It is not surprising, then, that as a high-school student Noller decided one day to drive up to the University of California at Berkeley, where he told a receptionist he wanted “to find out about biochemistry.” He was welcomed to the office of professor Donald McDonald, who spent an hour kindly satisfying his curiosity, thereby influencing his decision to major in biochemistry at Berkeley.
Noller graduated in 1960 and worked as a lab technician for a year before attending graduate school at the University of Oregon, where he trained as a protein chemist. After receiving his Ph.D. in 1965, Noller started a postdoc at the Medical Research Council Laboratory of Molecular Biology in Cambridge, U.K., determining the amino-acid sequence of glyceraldehyde-3-phosphate dehydrogenase. A year later he was doing a postdoc at the University of Geneva, working with Alfred Tissières on ribosomes. Noller’s new line of inquiry was fueled by a chance encounter with Sydney Brenner, also then at the MRC, at a tweed-and-sherry party. Brenner was part of the team in 1961 that genetically demonstrated the triplet nature of the translational code. At the party, Brenner said to Noller, “If you’re a protein chemist, why don’t you work on something interesting, like ribosomes?” Noller writes in his “Reflections” article that he realized “you can spend your life and career working on something boring or something exciting.” So he read up on ribosomes. In Tissières’ lab, Noller confirmed that the numerous bands isolated from the 30S and 50S ribosomal subunits were indeed different proteins; outside of the lab, Noller could be found playing jazz saxophone across Europe.
Noller became a faculty member at the University of California, Santa Cruz, in 1968, a time when the ribosome was known as a multienzyme protein complex, and the notion that RNA is enzymatic was downright preposterous.
At UCSC, “there were not a lot of ‘experts’ around to discourage you from going in unusual directions,” Noller writes. His lab tested modification reagents to knock out ribosome activity. Successful inactivation with Rose Bengal, which targets histidine in proteins and guanine in RNA, led Noller’s team to test a guanine-specific reagent, kethoxal, which left the ribosomal proteins active but the ribosome inactive. Additionally, the lab showed protection from kethoxal inactivation with prior tRNA binding. The lab quickly identified the kethoxal-modified guanines and spent the next decade identifying the tRNA-protected sites.
During this time, finding more than half of the published sequences incorrect, Noller came to terms with having to sequence rRNA. Multiple events influenced his approach. In 1975, Noller went on a three-part sabbatical to three different institutions. During the second leg, at the University of Geneva, he serendipitously ran into Joel Kirschbaum, who happened to have a λ transducing phage containing the entire rrnB operon and who taught Noller how to grow the virus and extract the DNA. During the third leg of his sabbatical, in Fred Sanger’s lab at the MRC in Cambridge, Noller learned DNA sequencing from Bart Barrell. Moreover, Wayne Barnes in the lab taught him to clone his DNA into ColE1 plasmid using restriction enzymes.
He recalls, “I had the eerie sensation that everything was falling into place guided by a mysterious force.” The final event was a “crucial conversation with Jürgen Brosius at a sidewalk café in Geneva.” As a result, Brosius came to do postdoctoral research with Noller and set up a system of running 16 sequencing gels a day. Noller’s team thus finished the 16S rRNA sequence, the sequence of the 23S rRNA and then all of the rrnB operon.
The team next determined rRNA secondary structure by sequencing several phylogenetically distinct 16S and 23S rRNAs. To this end, the lab purchased a Sun Microsystems workstation with an 86-MB hard drive and recruited an undergrad to develop a multiple-sequence alignment program. Carl Woese, at the University of Illinois at Urbana-Champaign, and Noller together used an approach they called “red dot-green dot” to visualize complementary sequences showing mirror-symmetric patterns of red transversions and green-dotted transitions.
Because the protein-centric view of ribosome function still pervaded, Noller’s group studied rRNA with little competition for a decade until the 1980s, when self-splicing introns were discovered. From the work of his students Danesh Moazed, Seth Stern and Ted Powers “came the hybrid-states mechanism for translocation, the placement of antibiotics in functional sites in the ribosomal RNA, and an initial model for the three-dimensional folding of 16S rRNA,” Noller notes. Jostled by future Nobel laureate Phil Sharp’s 1987 quip — “So, Harry, why don’t you nail it?” — Noller performed his seminal work, published in the journal Science, from whence the function of enzymatic rRNAs became widely accepted. He showed peptide bond formation from SDS-treated, SDS-and-proteinase-K-treated, and SDS-and-proteinase-K-treated and phenol-vortexed ribosomes.
Noller then moved on to a three-dimensional crystal structure of the entire ribosome with substrates mRNA and tRNAs in place. He recruited Jamie Cate as a postdoc and invited Marat and Gulnara Yusupov to UCSC from the CNRS lab in Strasbourg. Together they crystallized and phased the Thermus thermophilus 70S ribosome at 5.5-Å resolution. The initial lower resolution structures with and without bound tRNA helped in phasing and revealed an L-shaped tRNA in the ribosome A site. Across the continent and ocean, three other groups also solved the atomic structures of the ribosomal subunits. Noller writes, “Although at lower resolution, we could see the whole thing: how the subunits fitted together with their dozen intersubunit bridges; how the tRNAs bound to the A, P, and E sites of the ribosome; and the path of the mRNA through the ribosome. As we anticipated, all of the functional sites were made almost exclusively of ribosomal RNA.” Noller closes his “Reflections” article by alluding to the RNA world in a comment on the ongoing search for the secret of life, marveling at his fortune in being able to have a singular career focus.

Soo Hee LeeSoo Hee Lee ( received a Ph.D. in biochemistry from the Johns Hopkins University School of Medicine and undertook a Jane Coffin Childs Memorial Fund postdoctoral fellowship at the Yale University School of Public Health.

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