Alexander Spirin’s Vision of the Ribosome as a Thermal Ratchet Machine
Joachim Franka
Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, 2-221 Black Building, 650 W. 168th Street, New York, NY 10032, USAReceived May 13, 2021; Revised May 13, 2021; Accepted May 13, 2021
DOI: 10.1134/S0006297921080022
Alexander Spirin was a true visionary in the ribosome field. Among his many contributions is the one I wish to highlight, which is in an area of translation close to my heart: mRNA-tRNA translocation. This step in the ribosome’s work cycle is required to advance the mRNA, along with the tRNAs bound to it, by the space of one codon. Unlike the other steps in this cycle, this one involves a very large conformational change, the ratchet-like intersubunit rotation. Early on both Spirin [1, 2] and Mark Bretscher [3] independently proposed, well before any details of the ribosome’s structure were known, that translocation involves a relative motion between its two subunits, explaining the very rationale of the ribosome’s two-subunit architecture maintained in 3.5 billion years of evolution. While rotation is not explicitly mentioned in either of these papers, Bretscher writes as he proposes the hybrid model of translocation that “some relative movement of the two ribosomal subunits is suggested by the universal existence of two separable particles making up a 70S ribosome,” and Spirin suggests in a similar vein that the “periodical unlocking and locking of the subparticles of the ribosome is the driving mechanism providing for the displacements (translocations) of tRNAs, mRNA and peptidyl during translation.” This insight by two scientists working independently, going by the sparse structural information available at that time, was quite remarkable, and so is the story of how the details emerged later on, as eloquently recalled four decades later by Horan and Noller [4]. While Bretscher subsequently pursued other research aims, Spirin continued to further develop his ideas about the mechanism of translocation.
Alexander Spirin and Joachim Frank. Pushchino, Moscow Region, Russia. September 2011.
I must pause here to reflect on the fact that in 1968 I had just joined the lab of Walter Hoppe as a graduate student, trained as a physicist without any knowledge of biology, let alone the enigmas of the ribosome. Instead, I was starting to think about ways to process images from the electron microscope and about the ways to reconstruct molecules without having to rely on their being ordered in a crystal. This approach, proposed in 1975 [5], turned out to be of crucial importance in the study of molecules that act as molecular machines and constantly undergo changes in conformation.
Twenty-five years would go by after the initial conception, before we discovered first structural evidence for the intersubunit motion in my lab using single-particle cryo-electron microscopy [6]. By that time, this novel technique of structure research had advanced enough to depict ribosome architecture in great detail, down to the movable domains of the subunits and the locations of inter-subunit bridges. Also, by that time Bretscher’s hybrid model in which he postulated an intermediate step in translocation had been experimentally confirmed in Harry Noller’s lab [7].
In a nutshell, our cryo-EM results were as follows: comparison of the ribosome bound to EF-G in its GTP form with the ribosome bound to it in its GDP form showed an intersubunit rotation by about seven degrees, producing a displacement between the A sites of 30S and 50S subunits of roughly the amount expected for the advancement of the mRNA by one codon. Spirin celebrated the experimental validation of his early ideas in a minireview published in FEBS Letters [8]. In this article he brings up the concept of the ribosome as a molecular machine for the first time: “Instead of mechanical transmission mechanisms and power-stroke ‘motors’, thermal motion- and chemically-induced changes in affinities of ribosomal binding sites for their ligands (tRNAs, mRNA, elongation factors) are proposed to underlie all the directional movements within the ribosomal complex.” The seed for the idea that the ribosome is a Brownian thermal ratchet machine can be seen in his early discovery of “nonenzymic translocation” being stimulated by p-chloromercuribenzoate [9]; that is, the displacement necessary for translocation can apparently be reached by thermally driven intersubunit motion alone.
Again, Spirin was well ahead of his time since evidence for thermally driven intersubunit motion would not be found until five years later (counting from the FEBS Letters paper in 2002 [8]), through FRET spectroscopy – by solution FRET in the Noller’s lab [10] and later by single-molecule FRET in both Ruben Gonzalez’s [11] and Takjep Ha’s [12] labs. Finally, cryo-EM studies confirmed that in thermal equilibrium pretranslocational ribosomes exist in at least three conformations related by the ratchet-like intersubunit motion, including an intermediate conformation [13], just as would have been predicted by the hybrid model and the FRET studies.
Spirin’s most comprehensive view on the mechanism of translocation, informed by the evidence accumulated by that time, is contained in Ref. [14] and in a book chapter he co-wrote with Alexei Finkelstein on my invitation [15]. Since that time many studies looking into atomic detail have refined our understanding of translocation (e.g. [16]). But to me the emergence of structural and FRET evidence for a hypothesis conceived by Spirin’s ingenious intuition regarding mRNA-tRNA translocation, a universal process of life, is one of the great stories of science, and I’m glad to have been part of it.
Acknowledgments. This study was supported by grant 1R35 GM139453 from the National Institutes of Health to J. F.
Ethics declarations. The author declares no conflict of interest. This article does not contain description of studies with the involvement of humans or animal subjects.
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