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• W2080674156 abstract "The research group of Professor Andrey Belozersky with whom I started my academic career in 1955 consisted of two parts: one was located at the Department of Plant Biochemistry, Moscow State University, and the other at the A. N. Bach Institute of Biochemistry, Academy of Sciences of the USSR. This biochemical group was one of the most creative in the country. It was world-renowned because of several important discoveries in the field of nucleic acid studies. In the thirties of the last century, it succeeded in settling the question of the universal occurrence of two known types of nucleic acids, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), in living matter. At that time, many biochemists believed that RNA is a characteristic component of plants and fungi, whereas DNA (designated as “thymonucleic acid” or “animal nucleic acid”) belongs to the animal kingdom. The presence of DNA in plant cells raised doubts, as the positive cytochemical Feulgen reaction in plant cell nuclei was the only indirect evidence. Belozersky and colleagues were the first to isolate thymine and then DNA (thymonucleic acid) from higher plants (1Kiesel A. Beloserskii A. Hoppe-Seyler’s Z. Physiol. Chem. 1934; 229: 160-166Crossref Scopus (2) Google Scholar, 2Belozersky A.N. Dubrovskaya I.I. Biokhimiya. 1936; 1: 665-675Google Scholar), thus proving the universal occurrence of DNA. The next series of studies was carried out on bacteria (3Belozersky A.N. Mikrobiologiya. 1940; 9: 107-113Google Scholar) and demonstrated that both RNA and DNA were present there, again confirming the idea of the universality of the occurrence of both types of nucleic acids in organisms of different phylogenetic kingdoms. At the same time, the studies on bacteria showed that these organisms were deserving of special attention because of the high content of nucleic acids in their cells. During the years from 1939 to 1947, the systematic studies of the content of nucleic acids in bacteria of various taxonomic families, of different ages, and under different physiological conditions were performed in both subgroups headed by Belozersky (4Belozersky A.N. Cold Spring Harbor Symp. Quant. Biol. 1947; 12: 1-6Crossref Google Scholar). The high level of nucleic acids in cells was postulated to be in direct relation to their biological activities, growth rate, and cell proliferation. I joined the group in 1954 as a graduate student, formally at the Institute of Biochemistry of the Academy of Sciences, but the place of my experimental work was in the well equipped new building of the Biological Faculty at the Moscow State University. By that time, the Journal of Biological Chemistry had published a series of papers by Chargaff and colleagues in which the first convincing results that the base composition of nucleic acids can vary in different organisms were presented (5Vischer E. Chargaff E. J. Biol. Chem. 1948; 176 (715–734): 703-714Abstract Full Text PDF PubMed Google Scholar, 6Chargaff E. Vischer E. Doniger R. Green C. Misani F. J. Biol. Chem. 1949; 177: 405-416Abstract Full Text PDF PubMed Google Scholar, 7Vischer E. Zamenhof S. Chargaff E. J. Biol. Chem. 1949; 177: 429-438Abstract Full Text PDF PubMed Google Scholar, 8Chargaff E. Magasanik B. Vischer E. Green C. Doniger R. Elson D. J. Biol. Chem. 1950; 186: 51-67Abstract Full Text PDF PubMed Google Scholar). Crick and Watson had just published their famous papers on DNA structure and its implications for gene duplication and transcription into RNA (9Watson J.D. Crick F.H. Nature. 1953; 171: 737-738Crossref PubMed Scopus (7305) Google Scholar, 10Watson J.D. Crick F.H. Nature. 1953; 171: 964-967Crossref PubMed Scopus (1017) Google Scholar). The following questions had arisen. What is the range of variations of base compositions of DNA and RNA in different organisms? Does the total RNA just copy the total DNA of the cell, thus repeating its base composition, or do DNA-independent fractions of RNA exist? In 1956, I started work on testing the idea of the presumable correlation between the base compositions of RNA and DNA. The result was unexpected: the total DNA base composition manifested wide species variations, whereas the RNA composition was found to be surprisingly conserved (11Spirin A.S. Belozersky A.N. Shugaeva N.V. Vanyushin B.F. Biokhimiya. 1957; 22: 744-754PubMed Google Scholar). At the same time, statistical analysis of the data showed that a positive correlation of the base compositions of total RNAs of different species with their DNA compositions does exist, although at a low regression value (12Belozersky A.N. Spirin A.S. Nature. 1958; 182: 111-112Crossref PubMed Scopus (18) Google Scholar). The data were interpreted in such a way that a relatively small fraction of species-specific (i.e. gene-specific) RNA exists at the background of the main mass of evolutionarily conserved (presumably “non-genetic” or non-coding) RNA, which consists for the most part of ribosomal RNA. These results and interpretations were widely discussed in the literature and at conferences, in particular by F. H. Crick (13Crick F.H. Brookhaven Symp. Biol. 1959; 12: 35-39PubMed Google Scholar), F. Jacob and J. Monod (14Jacob F. Monod J. Cold Spring Harbor Symp. Quant. Biol. 1961; 26: 193-209Crossref Google Scholar), C. Levinthal (15Signer E.R. Torriani A. Levinthal C. Cold Spring Harbor Symp. Quant. Biol. 1961; 26: 31-34Crossref PubMed Google Scholar), S. Spiegelman (16Spiegelman S. Cold Spring Harbor Symp. Quant. Biol. 1961; 26: 75-90Crossref PubMed Google Scholar), and M. Yčas (17Yčas M. The Biological Code. North-Holland Publishing Co., Amsterdam1969Google Scholar). To illustrate the situation in those years, two citations are given below. The coding problem has so far passed through three phases. In the first, the vague phase, various suggestions were made, but none was sufficiently precise to admit disproof. The second phase, the optimistic phase, was initiated by Gamov in 1954, who was rash enough to suggest a fairly precise code. This stimulated a number of workers to show that his suggestions must be incorrect and, in doing so, increased somewhat the precision of thinking in this field. The third phase, the confused phase, was initiated by the paper of Belozersky and Spirin in 1958… The evidence presented there showed that our ideas were in some important respects too simple. (cited from Ref. 13Crick F.H. Brookhaven Symp. Biol. 1959; 12: 35-39PubMed Google Scholar, p. 35)It has long been believed that structural information was transferred from the genes to stable templates, such as ribosomal RNA, copied along the genes and maintaining in the cytoplasm the information necessary for protein synthesis. Every gene was supposed to determine the production of a particular type of ribosomal particles which in turn ensured the synthesis of a particular protein (see Crick, 1958 (18Crick F.H. Symp. Soc. Exp. Biol. 1958; 12: 138-163PubMed Google Scholar)). In recent years, however, this hypothesis has encountered several difficulties. 1. The diversity of base composition found in the DNA of different bacterial species is not reflected in the base composition of ribosomal RNA (Belozersky and Spirin, 1960). (cited from Ref. 14Jacob F. Monod J. Cold Spring Harbor Symp. Quant. Biol. 1961; 26: 193-209Crossref Google Scholar, p. 195) Thus, Belozersky and I found ourselves among the pioneers of messenger RNA studies. Our results were the first indications that only a small fraction of total RNA of normal (non-infected) cells copies DNA (genes) and, hence, could be supposed to play the role of messenger (as such an RNA was named by Jacob and Monod (19Jacob F. Monod J. J. Mol. Biol. 1961; 3: 318-356Crossref PubMed Google Scholar)) from DNA to proteins (i.e. to be a coding RNA). In 1962, Belozersky retired from his position as head of the laboratory at the Institute of Biochemistry, which I inherited. Together with my colleagues, we decided to move our investigations to the study of mRNA in eukaryotic (animal) cells. Using a new object, fish embryos, we made another discovery, that mRNA in eukaryotic cells does not exist in a free form, but, even when it is not engaged in translation, it is present in the form of messenger ribonucleoproteins (RNP particles) with a characteristic protein/RNA ratio of ∼3:1 (20Spirin A.S. Belitsina N.V. Ajtkhozhin M.A. J. Gen. Biol. 1964; 25 (English Translation (1965) Fed. Proc.24, T907–T915): 321-338Google Scholar, 21Spirin A.S. Eur. J. Biochem. 1969; 10: 20-35Crossref PubMed Google Scholar). These messenger RNP particles were named informosomes. In oogenesis and early embryogenesis, the RNP particles were proposed to be a masked form of mRNA (22Spirin A.S. Curr. Topics Dev. Biol. 1966; 1: 1-38Crossref PubMed Scopus (59) Google Scholar). Many years later, N. Standart, T. Hunt, and associates presented one of the most elegant experimental proofs of this proposal (23Standart N. Dale M. Stewart E. Hunt T. Genes Dev. 1990; 4: 2157-2168Crossref PubMed Google Scholar, 24Standart N. Hunt T. Enzyme. 1990; 44: 106-119Crossref PubMed Scopus (13) Google Scholar). Yet as the great bulk of the cellular RNA was implied to be a non-coding RNA (11Spirin A.S. Belozersky A.N. Shugaeva N.V. Vanyushin B.F. Biokhimiya. 1957; 22: 744-754PubMed Google Scholar, 12Belozersky A.N. Spirin A.S. Nature. 1958; 182: 111-112Crossref PubMed Scopus (18) Google Scholar), my interest was shifting to the structural and functional characteristics of this substance. As the RNA of ribosomes was already known to comprise at least 80% of the total RNA of a bacterial cell, it was quite evident that the major non-coding RNA should be ribosomal RNA. This expectation was confirmed by the analyses of base compositions of RNA-containing fractions of bacterial cells conducted by several groups (25Woese C.R. Nature. 1961; 189: 920-921Crossref PubMed Scopus (4) Google Scholar, 26Miura K.I. Biochim. Biophys. Acta. 1962; 55: 62-70Crossref PubMed Scopus (12) Google Scholar, 27Midgley J.E. Biochim. Biophys. Acta. 1962; 61: 513-525Google Scholar). Our first contribution to the understanding of ribosomal RNA was the demonstration that its high-molecular-weight molecules are constituted of a single covalently continuous polyribonucleotide chain each (28Spirin A.S. Milman L.S. Dok. Akad. Nauk SSSR. 1960; 134: 717-720Google Scholar, 29Spirin A.S. Biokhimiya. 1961; 26: 511-522Google Scholar, 30Spirin A.S. Ebel J.-P. Grunberg-Manago M. Acides Ribonucléiques et Polyphosphates: Structure, Synthèse et Fonctions, Colloques Intrernationaux CNRS No. 106, Strasbourg, 6–12 Juillet 1961. Édition du CNRS, Paris1962: 75-87Google Scholar, 31Bogdanova E.S. Gavrilova L.P. Dvorkin G.A. Kisselev N.A. Spirin A.S. Biokhimiya. 1962; 27: 387-402PubMed Google Scholar) but are not composed of smaller RNA subunits, as had been assumed previously (32Hall B.D. Doty P. J. Mol. Biol. 1959; 1: 111-126Crossref Google Scholar, 33Takanami M. Biochim. Biophys. Acta. 1960; 39: 152-154Crossref PubMed Scopus (0) Google Scholar, 34Brown R.A. Ellem K.A. Colter J.S. Nature. 1960; 187: 509-511Crossref Scopus (2) Google Scholar, 35Aronson A.I. McCarthy B.J. Biophys. J. 1961; 1: 215-226Abstract Full Text PDF PubMed Google Scholar). The discovery of the self-folding of the high-polymer polyribonucleotide chains into specific compact globular bodies was the principal achievement that attracted us to further studies of ribosomes. First, it was demonstrated that the conformation of a high-polymer RNA can change from the state of an unfolded flexible chain in the absence of Mg2+ at low ionic strength to the state of more compact rod-like particles, still flexible but possessing a developed secondary structure in the presence of Mg2+ at moderate ionic strength, and further to the state of well shaped compact globules at elevated Mg2+ concentrations and ionic strengths (Refs. 36Spirin A.S. J. Mol. Biol. 1960; 2: 436-446Crossref Google Scholar, 37Kisselev N.A. Gavrilova L.P. Spirin A.S. J. Mol. Biol. 1961; 3: 778-783Crossref PubMed Google Scholar, 38Spirin A.S. Prog. Nucleic Acid Res. 1963; 1: 301-345Crossref Scopus (113) Google Scholar; see also Refs. 30Spirin A.S. Ebel J.-P. Grunberg-Manago M. Acides Ribonucléiques et Polyphosphates: Structure, Synthèse et Fonctions, Colloques Intrernationaux CNRS No. 106, Strasbourg, 6–12 Juillet 1961. Édition du CNRS, Paris1962: 75-87Google Scholar and 31Bogdanova E.S. Gavrilova L.P. Dvorkin G.A. Kisselev N.A. Spirin A.S. Biokhimiya. 1962; 27: 387-402PubMed Google Scholar). Later, my colleague V. D. Vasiliev and associates showed that electron microscopy images of two species of isolated ribosomal RNA (16 S and 23 S) in the compactly folded state are different in their shapes and strongly resemble the images of isolated 30 S and 50 S ribosomal subunits, respectively (39Vasiliev V.D. Selivanova O.M. Koteliansky V.E. FEBS Lett. 1978; 95: 273-276Crossref PubMed Scopus (36) Google Scholar, 40Vasiliev V.D. Zalite O.M. FEBS Lett. 1980; 121: 101-104Crossref PubMed Scopus (16) Google Scholar). This led us to boldly assert that the specific shape and gross structure of ribosomal particles are determined by self-folding of their high-polymer ribosomal RNAs (41Vasiliev V.D. Serdyuk I.N. Gudkov A.T. Spirin A.S. Hardesty B. Kramer G. Structure, Function, and Genetics of Ribosomes. Springer-Verlag New York Inc., New York1986: 128-142Google Scholar). More recently, this assertion was confirmed by direct x-ray structural analyses of ribosomes (“The shape [of the 30S ribosomal particle] is largely determined by the RNA component; none of the gross morphological features is all protein.” (cited from Ref. 42Wimberly B.T. Brodersen D.E. Clemons Jr., W.M. Morgan-Warren R.J. Carter A.P. Vonrhein C. Hartsch T. Ramakrishnan V. Nature. 2000; 407: 327-339Crossref PubMed Scopus (1630) Google Scholar)). Thus, ribosomal RNA could be considered as the structural core of ribosomal particles. In addition to the structure-forming capacity of ribosomal RNA, the high conformational mobility of the folded RNA depending on ionic conditions, temperature, and the presence of some solutes seemed to be an intriguing property of the RNA in light of its possible functional role in the ribosome (43Spirin A.S. Macromolecular Structure of Ribonucleic Acids. Reinhold Publishing Corp., New York1964Google Scholar). When compact ribosomal particles were exposed to the same physical and chemical conditions that were used in the RNA studies, they exhibited a similar conformational response. Depletion of Mg2+ caused stepwise unfolding of ribosomal subunits through several discrete intermediate states without loss of ribosomal proteins, thus demonstrating the scaffold role of ribosomal RNA in the ribosome structure, on one hand, and the possibility of conformational mobility of ribosomal particles without their destruction, on the other (44Spirin A.S. Kiselev N.A. Shakulov R.S. Bogdanov A.A. Biokhimiya. 1963; 28: 920-930PubMed Google Scholar, 45Gavrilova L.P. Ivanov D.A. Spirin A.S. J. Mol. Biol. 1966; 16: 473-489Crossref PubMed Google Scholar). Another type of reversible structural transformation of ribosomal particles in vitro was shown upon their exposure to high ionic strength in the presence of Mg2+ (46Spirin A.S. Belitsina N.V. Lerman M.I. J. Mol. Biol. 1965; 14: 611-615Crossref PubMed Google Scholar, 47Lerman M.I. Spirin A.S. Gavrilova L.P. Golov V.F. J. Mol. Biol. 1966; 15: 268-281Crossref PubMed Google Scholar). Under these conditions, ribosomal proteins dissociated from ribosomal RNA in a stepwise manner while the compactness of the particles and their gross morphology remained the same (see also Ref. 41Vasiliev V.D. Serdyuk I.N. Gudkov A.T. Spirin A.S. Hardesty B. Kramer G. Structure, Function, and Genetics of Ribosomes. Springer-Verlag New York Inc., New York1986: 128-142Google Scholar). This stepwise disassembly of compact ribosomal particles was found to be reversible, with restoration of their biological activity (47Lerman M.I. Spirin A.S. Gavrilova L.P. Golov V.F. J. Mol. Biol. 1966; 15: 268-281Crossref PubMed Google Scholar, 48Spirin A.S. Belitsina N.V. J. Mol. Biol. 1966; 15: 282-283Crossref PubMed Google Scholar). The experiments on successful reassembly (reconstitution) of biologically active ribosomal particles were simultaneously published by the groups of M. Nomura (49Hosokawa K. Fujimura R.K. Nomura M. Proc. Natl. Acad. Sci. U.S.A. 1966; 55: 198-204Crossref PubMed Google Scholar) and M. Meselson (50Staehelin T. Meselson M. J. Mol. Biol. 1966; 16: 245-249Crossref PubMed Google Scholar). (It is noteworthy that 3 years before, preliminary results on the in vitro assembly of ribosome-like particles from ribosomal RNA-containing “CM-particles” and cell lysate proteins were obtained and reported at the Cold Spring Harbor Symposium (51Spirin A.S. Cold Spring Harbor Symp. Quant. Biol. 1963; 28: 267-268Crossref Google Scholar).) The function of the ribosome is to translate the genetic information encoded in the nucleotide sequences of mRNA into amino acid sequences of polypeptide chains of proteins. During the process of translation, the ribosome performs the unidirectional driving of tRNA macromolecules through itself and the coupled drawing of the mRNA chain from its 5′- to 3′-end. In the course of translation, the free energies of the transpeptidation reaction and the GTP hydrolysis reaction are consumed (Fig. 1). Thus, the translating ribosome can be considered as a conveying molecular machine, simultaneously being a “technological” protein-synthesizing machine. Obviously, the ribosome as a conveying machine must be capable of performing its own mechanical movements. More than 2 decades ago, it was proposed that the functional movements of the translating ribosome are based on the overall construction of the ribosome, allowing certain anisotropic motions generated by thermal Brownian movements of large blocks of the ribosome and the ribosomal subunits (52Spirin A.S. Prog. Nucleic Acids Res. Mol. Biol. 1985; 32: 75-114Crossref PubMed Scopus (87) Google Scholar). These ideas were further developed in subsequent publications (53Spirin A.S. Kleinkauf H. von Dören H. Jaenicke R. The Roots of Modern Biochemistry. Walter de Gruyter & Co., Berlin1988: 511-533Google Scholar, 54Spirin A.S. FEBS Lett. 2002; 514: 2-10Crossref PubMed Scopus (62) Google Scholar, 55Spirin A.S. RNA Biol. 2004; 1: 3-9Crossref PubMed Google Scholar). The fact that ribosomes are universally built from two loosely associated and easily separable subunits in all living beings is one of the most fascinating properties of the translation machinery. The two subunits (Fig. 2), the small one (the so-called 30 S in prokaryotes or 40 S in eukaryotes) and the large one (50 S in prokaryotes or 60 S in eukaryotes), have different functions: the small subunit is responsible for the “genetic” functions of the ribosome, such as binding of mRNA and decoding of genetic information, whereas the large subunit acts as its “catalytic” partner, being responsible for the formation of peptide bonds and the attraction of protein catalysts for GTP hydrolysis. Thus, a clear division of labor exists between the two ribosomal subunits. It is remarkable that none of the subunits alone is capable of performing the coupled unidirectional movement of mRNA and tRNA, the conveying function designated as translocation. On the basis of the above knowledge, I proposed that (i) the main functional purpose of the two-subunit construction of the ribosome is the organization of the translocation mechanism of the ribosome, (ii) translocation requires mutual mobility of the ribosomal subunits, and (iii) translocation proceeds through an intermediate state when the products of the transpeptidation reaction (peptidyl-tRNA and deacylated tRNA) occupy positions with shifted 3′-ends of tRNAs on the large subunit but yet non-shifted codon-anticodon duplexes on the small subunit (56Spirin A.S. Dok. Akad. Nauk SSSR. 1968; 179: 1467-1470PubMed Google Scholar, 57Spirin A.S. Curr. Mod. Biol. 1968; 2: 115-127PubMed Google Scholar, 58Spirin A.S. Cold Spring Harbor Symp. Quant. Biol. 1969; 34: 197-207Crossref PubMed Google Scholar). Similar ideas were published at the same time by M. S. Bretscher (59Bretscher M.S. Nature. 1968; 218: 675-677Crossref PubMed Scopus (98) Google Scholar), although the two models differed in detail. The mechanistic principle of my model was based upon the idea that the associated subunits of the translating ribosome pass through the stage of an “unlocked” (open) ribosome. Thus, the ribosome was considered as a particle oscillating between “locked” (closed) and unlocked (open) conformations. The unlocked states were proposed to be required both at the aminoacyl-tRNA binding step to allow the large substrate (aminoacyl-tRNA) to enter into the intersubunit space of the ribosome and at the translocation step to facilitate the products’ movement inside the ribosome (peptidyl-tRNA) and exit from the ribosome (deacylated tRNA). Experimental testing of the hypothesis was delayed, however, for many following years because of the lack of adequate methodologies to study the dynamics of macromolecular complexes. Nevertheless, the attempts to detect macroconformational changes within ribosomes during translation were undertaken from time to time. The first experimental evidence in favor of intraribosomal conformational mobility of the translating ribosome came from comparison of the compactness of the particles before and after translocation. It was shown by sedimentation analysis that the sedimentation coefficient of the post-translocation ribosome is somewhat less than that of the pre-translocation ribosome (the difference was ∼1 S) (60Baranov V.I. Belitsina N.V. Spirin A.S. Methods Enzymol. 1979; 59: 382-397Crossref PubMed Scopus (8) Google Scholar). However, this fact could be explained either by changing the composition of the ribosome as a result of translocation (loss of the deacylated tRNA molecule) or by a conformational change, such as some “swelling” of one of the ribosomal subunits or of the ribosome as a whole (the latter could result from a widening of the intersubunit space). Neutron scattering in various mixtures of H2O and D2O allowed these alternatives to be distinguished. The point is that the RNA component of the ribosome becomes contrast-matched, i.e. “invisible” for neutrons, in a solvent with a proper proportion of light and heavy water (70% D2O). This enabled the measurement of compactness (radius of gyration (Rg)) of only the protein component of the ribosomal particle, irrespective of the number of bound tRNA molecules. It was found that the Rg of the protein component of the post-translocation ribosome was somewhat greater than that of the pre-translocation ribosome (ΔRg = 1–3 Å) (61Serdyuk I.N. Spirin A.S. Hardesty B. Kramer G. Structure, Function, and Genetics of Ribosomes. Springer-Verlag New York Inc., New York1986: 425-437Google Scholar, 62Spirin A.S. Baranov V.I. Polubesov G.S. Serdyuk I.N. May R.P. J. Mol. Biol. 1987; 194: 119-126Crossref PubMed Google Scholar). In other words, translocation made the whole ribosome slightly less compact. These experiments were the first physical evidence of a conformational change in the translating ribosome as a result of translocation. However, they did not answer the question of whether the slight decrease in ribosome compactness upon translocation reflects an intersubunit change or a conformational alteration within one of the subunits. Further neutron-scattering experiments, including those with selectively deuterated ribosomal subunits (either 30 S or 50 S), led to the conclusion that conformational changes within the small subunit made a major contribution to the effect of the increase in the Rg of the full ribosome upon translocation (see “Intrasubunit Large-block Mobility”) (61Serdyuk I.N. Spirin A.S. Hardesty B. Kramer G. Structure, Function, and Genetics of Ribosomes. Springer-Verlag New York Inc., New York1986: 425-437Google Scholar, 63Serdyuk I. Baranov V. Tsalkova T. Gulyamova D. Pavlov M. Spirin A. May R. Biochimie. 1992; 74: 299-306Crossref PubMed Scopus (32) Google Scholar). Recent developments in the cryoelectron microscopy technique allowed J. Frank and colleagues to demonstrate a real intersubunit movement coupled with translocation: they detected a rotational shift of one ribosomal subunit relative to the other around an axis perpendicular to the subunit interface (64Frank J. Agrawal R.K. Nature. 2000; 406: 318-322Crossref PubMed Scopus (632) Google Scholar, 65Valle M. Zavialov A. Sengupta J. Rawat U. Ehrenberg M. Frank J. Cell. 2003; 114: 123-134Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). This rotation of the small subunit relative to the large subunit was estimated to be ∼6° counterclockwise if viewed from the small subunit. The rotation was accompanied by a widening of the intersubunit mRNA channel (64Frank J. Agrawal R.K. Nature. 2000; 406: 318-322Crossref PubMed Scopus (632) Google Scholar). The observation of such a rotation was confirmed in the studies by H. F. Noller’s group using a cross-linking technique, fluorescence resonance energy transfer (FRET) methodology, and “translation-libration-screw” (TLS) crystallographic refinement (66Horan L.H. Noller H.F. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 4881-4885Crossref PubMed Scopus (0) Google Scholar, 67Ermolenko D.N. Majumdar Z.K. Hickerson R.P. Spiegel P.C. Clegg R.M. Noller H.F. J. Mol. Biol. 2007; 370: 530-540Crossref PubMed Scopus (135) Google Scholar, 68Korostelev A. Noller H.F. J. Mol. Biol. 2007; 373: 1058-1070Crossref PubMed Scopus (25) Google Scholar). The ribosome was shown to be fixed (locked) in the rotated form upon binding of elongation factor (EF)-G, the catalyst of translocation, until EF-G and deacylated tRNA were released from the ribosome (64Frank J. Agrawal R.K. Nature. 2000; 406: 318-322Crossref PubMed Scopus (632) Google Scholar, 69Spiegel P.C. Ermolenko D.N. Noller H.F. RNA. 2007; 13: 1473-1482Crossref PubMed Scopus (99) Google Scholar). More recently, using single-molecule FRET methodology, it was found that ribosomes undergo spontaneous intersubunit movements oscillating between the original (“classical”) and rotated forms, with the equilibrium shifted toward either the original or rotated forms depending on the functional state of the ribosome (70Cornish P.V. Ermolenko D.N. Noller H.F. Ha T. Mol. Cell. 2008; 30: 578-588Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The following conclusions can be made from the recent FRET data. (i) Vacant ribosomes thermally oscillate between the original and rotated forms with relatively low forward and reverse rotation rates; the equilibrium is somewhat shifted toward the original form (the proportion of the two forms in the equilibrium mixture is ∼3:2). (ii) The binding of N-acylated aminoacyl-tRNA to the P site somewhat reduces the forward rotation rate and correspondingly slightly shifts the equilibrium toward the original (non-rotated) form, but still the proportion of the rotated form in the equilibrium mixture may be significant (up to one-third). (iii) The occupancy of the A site by N-acylated aminoacyl-tRNA and the P site by deacylated tRNA, which models the translating ribosome after transpeptidation, induces rapid oscillation of ribosomes between the original and rotated forms, with the equilibrium shifted to the rotated form; this state should correspond to the pre-translocation state ribosome. (iv) The binding of the translocation catalyst EF-G with a non-hydrolysable GTP analogue, when deacylated tRNA still remains in the P site (the situation that simulates the first step of translocation), fixes the rotated form of the ribosome. (v) The transition to the final post-translocation state, after GTP hydrolysis and the release of EF-G and deacylated tRNA, when peptidyl-tRNA occupies the P site and the A site becomes vacant, to some extent restores the situation mentioned in Conclusion (ii), but with somewhat higher rates of both the forward and reverse reactions. Thus, both pre-translocation and post-translocation state ribosomes in the absence of elongation factors oscillate between the original (classical) and rotated forms. The presence of deacylated tRNA in the P site after transpeptidation strongly stimulates the rates of both forward and, to a less extent, reverse rotational shifts of ribosomal subunits, shifting the equilibrium toward the rotated form. The binding of EF-G fixes the rotated form. The properties of the rotated form of the ribosome, such as the high rate of oscillation between the alternative conformations (in the absence of EF-G), the permissibility of translocational intraribosomal shifts of peptidyl-tRNA and deacylated tRNA, and the competence to accept EF-G as a translocation catalyst (64Frank J. Agrawal R.K. Nature. 2000; 406: 318-322Crossref PubMed Scopus (632) Google Scholar, 65Valle M. Zavialov A. Sengupta J. Rawat U. Ehrenberg M. Frank J. Cell. 2003; 114: 123-134Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 69Spiegel P.C. Ermolenko D.N. Noller H.F. RNA. 2007; 13: 1473-1482Crossref PubMed Scopus (99) Google Scholar), as well as the widening of the intersubunit mRNA channel (64Frank J. Agrawal R.K. Nature. 2000; 406: 318-322Crossref PubMed Scopus (632) Google Scholar), imply that the rotated form is equivalent to the unlocked state proposed earlier for the hypothetical intermediate in ribosomal translocation (56Spirin A.S. Dok. Akad. Nauk SSSR. 1968; 179: 1467-1470PubMed Google Scholar, 57Spirin A.S. Curr. Mod. Biol. 1968; 2: 115-127PubMed Google Scholar, 58Spirin A.S. Cold Spring Harbor Symp. Quant. Biol. 1969; 34: 197-207Crossref PubMed Google Scholar). It is noteworthy that more than 2 decades ago, base" @default.
• W2080674156 created "2016-06-24" @default.
• W2080674156 creator A5015282605 @default.
• W2080674156 date "2009-08-01" @default.
• W2080674156 modified "2023-09-29" @default.
• W2080674156 title "The Ribosome as a Conveying Thermal Ratchet Machine" @default.
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