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Abiogenic evolution of ribonucleic acid as a left-handed helix and its protecting substances on primordial earth – A story about evolution of ribosome and the origin of life

Kozo Nagano

Nagano Research Institute of Molecular Biology, 4-8-24 Higiriyama, Kohnan-ku, Yokohama 233-0015, Japan

DOI: 10.15761/IMM.1000284

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Abstract

Origin of life on earth must be started from a story explaining how ribonucleic acids (RNAs) had been accumulated in azoic era. Since the world of nonazoic era could have been full of water, a single molecule of RNA should have been in danger of hydrolysis rather than in a tendency of progressive synthesis. It necessarily requires coevolution of proteins that had been playing a role of protecting RNA from hydrolysis. The present article describes a possibility of a large-scale of confined organic phosphates and methane hydrates under high pressure and temperature deeply hidden into the mantle of the earth. In such conditions, a polymeric form of D-ribose can be made as a left-handed helix connected by base pairs with neighboring strands. In the lattice of left-handed helices of RNA, a lot of spaces or gaps allowed accumulation of various amino acids, from the smallest one such as glycine to not very large as tryptophan. At the 3’ end of a polymeric RNA strand, aminoacylation could also occur. The other essential substances to the origin of life, such as adenine-triphosphates, could have been accumulated together.

Introduction

Fossils have shown that biochemically advanced microbial life had existed on earth over 3 billion years ago. Since the age of earth is believed to be about 4.6 billion years, it would be natural to think that prebiotic molecules of nucleic acids and proteins had been accumulated either shortly after or at the same time of the birth of the earth, the surface of which at that time was magmatic ocean covered by a thick layer of vaporized water molecules and various gaseous molecules, rather rich in hydrogen molecules. Glycine, α-amino-isobutyric acid, alanine, glutamic acid, β-alanine, isovaline, aspartic acid, and leucine were observed in the Murchison meteorite [1]. Although they reported the relative abundance of L-enantiomer, it might not be the cause of the origine of the prevailance of L-enantiomer on the present-day earth [2,3]. In the Yamato-91198 meteorite, serine, threonine, proline, valine, isoleucine, sarcosine, α-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid, α-aminoisobutyric acid, α-aminoadiopic acid, various aliphatic carbonic acid, and aliphatic as well as aromatic hydrocarbons were observed. However, no sign of nucleic acids has been observed in meteorites except some purines and pyrimidines. Some of them might have been synthesized by a shock of thunder, while some of them, such as nucleic acids in particular, might have been destroyed on a boiled magmatic ocean. Accordingly, it remains an etigma how nucleic acid had been formed using D-riboses as its main component among many other pentose candidates. The present article tries to present a hypothesis that solves the problem of multiple difficulties to reach the origin of life from the origin of earth.

Evolution of earth

The origin of earth has been supposed to be a gathering center of meteorites and comets. A core of comet is usually composed of ice water and dry ice (carbon dioxide) as well as methane, ammonia, silicates, and phosphates. On the other hand, the components of meteorites are mainly reduced metals and metal oxides. Surface temperature of primordial earth would have been pretty high by thermal energy produced by collisions of comets and meteorites, but would have been cooled down when its core became occupied by components of meteorites and its surface was covered by those from comets. The above process in evolution of earth should be referred to as the first stage. The second stage in the history of earth started from temperature elevation in its core by accumulation of thermal energy originated from disintegration of uranium atoms. At this stage, the surface temperature would have been as cool as that of comets. As temperature in the core of the earth was elevated, water vapour would have been concentrated in the atmosphere of the ancient earth, while its surface would have been rather a magmatic ocean composed of exposed hot mantle, on which hot rain would have been falling to make a lot of boiling lakes. The boiling lakes might have grown up to form a boiling ocean. It should be referred to as the third stage. The fourth stage in the history of earth would be in the cooling process. When the first signs of the ancient life existed over 3 billion years ago, the evolution of RNA had already reached a ripening period. The basic hypothesis adopted in the present article is that the evolution of RNA started from the end of the first stage and continued until the end of the third stage.

Accumulation of organic phosphates and aromatic amines

A mineralogical form of the mixture of water and methane at a very low temperature under a high pressure would have been that of methane hydrate that is known to be very inflammable in contact with oxygen gas. This phenomenon would mean that methyl radical is easily formed by reacting with an oxygen atom. Since the primeval atmosphere is supposed to be rich in hydrogen molecules, the most of the reactive oxygen atoms would have been reserved in a form of ferrous oxide brought about by meteorites. Accordingly, one of the conceivable starting events of abiogenic biological substance synthesis would have been a contact of the methane hydrate and ferrous oxide in the deep center of the gaseous earth under a high pressure and a low temperature. Disintegration of uranium atoms might have played a role in producing oxygen radical, which might also be an origin of the formation of hot mantle and core of the earth by concentration of melted reduced iron in the center of the earth. The possible various chain reactions from the methyl radical would have produced various aliphatic and aromatic chains and fatty acids. Specific gravities of such organic compounds synthesized abiogenically on the primeval earth are supposed to be heavier than that of methane hydrate that carries much higher content of hydrogen atoms. Most of organic compounds brought about by meteorites would have been mixed with the newly synthesized ones. Those were precipitated deeply in order of specific gravity at the bottom layer on the mantle of the earth. Since the mantle were not well separated enough from the core, those organic compounds of approximately uniform composition had an intimate contact with the rocks of metallic meteorites containing ferrous oxide and uranium as well as silicates, sulfates, and phosphates. The first organic compound synthesized in this way would have been methanol, which could have occupied the lattice position of water molecule in the crystalline form of methane hydrate. Condensation reaction between two methanol molecules with an oxygen radical would have produced a molecule of diose that is a simplest form of sugar containing two carbon atoms. A similar condensation between one molecule of diose and methanol would have produced a triose without isomers. This is called glycerol. Phosphorylation of glycerol produce glycerophosphoric acid, which has three isomers. Triose molecules were synthesized just below the interface with the methane hydrate layer, but deposited deeply in the bottom of the mantle. Further evolution of organic compounds from triose could be independent of the presence of the methane hydrate. It is, however, important to assume that the main chemical reactions were promoted by oxygen radicals. A similar condensation reaction between two trioses could have produced hexoses, and a simultaneous condensation of three trioses with two oxygen radicals would have produced glycerols. Such a mixture of hexoses and glycerols could have made a contact with an energy-rich polyphosphoric acids in the rocks. Polyphosphates could also be formed inside a rock probably by intervention of oxygen radicals. It would have been a considerable period before accumulation of phosphohexoses and phosphoglycerols were completed. Such reactions could have proceeded at a low temperature. As the temperature of the mantle started elevating, dehydration reactions between phosphohexoses and between phosphoglycerols and fatty acids gradually started syntheses of polyphosphohexoses and phospholipids, both of which were separated from each other in accordance with their physicochemical characters.

Accumulation of phospho-D-ribose polymer as left-handed single-stranded helices

One of the points in the present hypothesis is that the primeval earth would have been covered by aliphatic and aromatic compounds including their derivatives such as fatty acids, because the densities of nitrogen-, sulfur-, and phosphorus-containing organic compounds are supposed to be larger than those containing only hydrogen, carbon and oxygen atoms. Accordingly, various amino acids without phosphorus atom would have been accumulated under the first layer of petroleum oil and fatty acids. The hydrogen-bonding network of methane hydrate would have been in the second layer mixed with ammonia, sulfate (or mercaptan), phosphin, amino acids, alkylamines, etc., and made a direct contact with ferrous oxide and silicates. Methanol radical as a reaction product of methyl radical and water would have been trapped inside the hydrogen-bonding network and would have resulted in formation of glyceride and phosphoglyceride. Glyceride would have been a starting material for various sugar synthesis including various pentoses. The reaction between phosphoglyceride and fatty acids would have formed a phospholipid as a single layer between the first petroleum oil layer and the second or third layers rich in hydrogen-bonding capacities. Accumulation of polyphosphate might have been important at this stage, because the leading role of organic reactions in evolution of both earrth and life would be shifting gradually from the radical reaction described above to polymerization by dehydration of amino acids and phosphosugars through reaction intermediates such as sugar triphosphates.

It is natural to suppose that the high concentration of water vapor kept the surface temperature not too high from that of boiling water. If the temperature was not too high, there could be enough amount of organic compounds covering the surface of the magmatic ocean, where polymerization of small organic compounds by dehydration reaction would have been prevailing, phosphorylation of polymerized organic compounds could have been processed in the melted solution of phosphate and silicate on the magma. The reason why sulfur atoms were not involved in the process of the polymerization reaction could be due to the boiling and destructive temperature for sulfurylated compounds compared to that of phosphorylated compounds as well as to the delicately adjusted surface temperature of the earth. It is, however, important to note that a primitive sulfur-oxidizing bacteria was found at submarine thermal springs on the Galapagos rift [4]. It utilizes chemosynthesis to derive entire energy supply from reactions between the sea water and the rocks at high temperatures, rather than photosynthesis. At the era of Origins of Life, sulfur oxidizing bacteria could have played a big role, but Corliss, et al. (1979) did not described anything about how the ancestral bacteria obtained energy-rich phosphate compounds and amino acids from the hot spring water. There could be a possibility of sulfur-containing RNA instead of normal phosphor-containing RNA. As described in the preceding section, accumulation of phosphoglyceride must have occurred under the petroleum oil and phospholipids layers before evolution of RNA. The reaction between phosphoglyceride and methyl radical in the methane hydrate network by oxygen atoms supplied from ferrous oxides would have produced various phosphorylated pentoses and hexoses. Water molecules in the hydrogen-bonding network would be replaced by those phosphorylated sugars as well as various amino acids and alkylamines. On the other hand, Martin, Baross, Kelley and Russell [5] found that the chemistry of the H2-CO2 redox couple in hydrothermal vents showed a striking parallelism with that of the core energy metabolic reactions of some modern prokaryotic autotrophs. Thus, there could be another question about what mechanism initiated the chemistry of life in such an environment as the hydrothermal vent areas. The basic hypothesis of the present article tries to throw a light on that point.

Condensation of glycerophosphoric acid with diose leads to various isomers of ribose, among which phosphor-D-ribose is the most prospective. On the other hand, condensation reactions with carbon radicals and ammonia could have accumulated aromatic ring compounds such as uracil, thymidyl, cytocyl, adenyl, guanosyl radicals etc, among which some hydroxyl ring compounds such as dihydrouracyl would have been included. Joyce (1989) has shown various courses of producing adenine, diaminopurine, hypoxanthine, guanine, and xanthine, starting from hydrogen cyanide (HCN), and passing through diaminomaleonitrile (DAMN) [6]. He has also shown that condensation of glycoaldehyde (HC=OCH2OH) with hydrogen carbon monoxide (H2CO) produces aldotetrose (tetrose), ribulose, xylulose, ribose, arabinose, xylose, lyxose (these are pentoses), and psicose, fructose, sorbose, tagalose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose (these are hexoses). The possible reactions of the phospho-D-ribose with uracil radical, which is the most predominant species among the aromatic compounds mentioned above, could have reached the goal of uridine. One of the biggest merit of 5’-phospho-D-ribose to the other conceivable sugars and sugar phosphates is that it can geometrically be extended infinitely as a left-handed single-stranded helix. The uracil bases planted at C1’ position allows a considerable stability for hydrogen-bonding capability with the neighboring left-handed single-stranded helices. Even if ideal Watson-Crick base pairs of adenine-uracil (A-U) and guanine-cytosine (G-C) or a wobble pair of guanine-uracil (G-U) are not available, uracil-uracil (U-U) base pair guarantees a relative stability. Since some tRNA molecules of animaria mithocondria contains a considerable amount of U-U base pairs in their double stranded helical regions [7]. The reason why the present author emphasizes very much about uridine 5’-phosphate is that it could have a possibility of infinitely large lattice formation with hydrogen-bonded U-U base pairs and left-handed single-stranded helices shown in Figure 1. The distance between two uracil bases is 7.2 Å.

If this is 3.6Å, which is usually the best distance for base stacking, the ring of D-ribose has a fatal near-contact with the neighboring D-ribose ring at 士6th position on the left-handed single-stranded helix. In that sense, the distance of 7.2 Å between the two uracil bases would mean that there is a possibility of intercalate another aromatic bases such as adenine, guanine, etc, between the layer of uracil-uracil hydrogen-bonding network. Figure 2 shows a part of such an infinite helix surrounded by three similar infinite helices stabilized by making U-U base pairs. Such a network of polyuridine chain protects itself from being hydrolyzed by hot water. Figure 3 shows that adenine triphosphate (ATP) can be stacked between the layer of uracil bases. Similar stacking of guanine triphosphate (GTP), and the other nucleotide polyphosphates could occur in the cavity to save the material, even though simple polymerization of such materials are not favorable in order to grow up the polyuridine layer. Joyce (1989) listed up the coenzymes in the present-day living organisms that resemble nucleotides, such as UDP-glucose, CDP-diacylglycerol, S-adenosylmethionine, flavin mononucleotide, thiamine pyrophosphate, nicotinamide, , etc. Those are all capable of being saved in the cavity [6]. The components of cell membranes such as phospholipids are also the competent candidates to be saved in similar way. Even amino acids are capable of being important bulking materials if their side chains are not too big. Polymerization of such small amino acids could have occurred by simple dehydration reaction. Figure 4 shows a case of L-amino acids polymerization growing up in the hole of cavity, even if it is not a right-handed α–helix.

The layers of the 6-fold symmetrical left-handed helices of polyuridines cannot be estimated about their thickness. Their outside surface would have been in contact with hot water vapor. Accordingly, it would have been covered by a thick skin of layers rich in phospholipids just like a big cell membrane, below which another thick layer of methane hydrates, ammonia, D-, and L-amino acids, and the other components in a state of chaotic mixture. The movement of the outside skin area would be supposed to be so violent that any regular sophisticated structure such as anticodon loop could not have a chance to evolve. In contrast, another contact surface with mantle, which contain crystalline structures of silicate, metal oxides and also variety of concave and convex structure, would have fitted to grow a special structure of polyuridines. Figure 5 shows that an amino acid glycine, which is drawn in pink, bonded with its carboxyl end to 3’-end of polyuridine at its bottom region of the left-handed single-stranded helix. This is an ancestral form of transfer RNA (tRNA) and is going to polymerize with proline for further polymerization of L-amino acids. N-terminal end of glycine in pink is bonded to carboxyl end of proline in blue. This kind of stereo-specific chemical reaction could never have evolved on the outside surface in contact with hot water but was possible on the surface of silicates. Figure 6 also shows that the largest side chain of tryptophan among the essential 20 kinds of L-amino acids could be accommodated in this environment. As the temperature and the pressure gradually lowered at the bottom of the polyuridine layers in contact with the mantle rocks, the aminoacylation between 3’-end of uridine and the carboxyl end of amino acids became harder without a supply of energy transfer from the reaction of ATP to ADP + H3PO4. Figure 7 shows a similar state to that of Figure 8 but ATP not yet inserted, while Figure 8 such a change by inserting the structure of ATP between the transition state of dipeptide Gly-Pro bound at 3’-O of polyuridine terminus on the long helix in brown which is separated from the other part of long helix (just one turn) in yellow. The nucleotides in magenta is a part of anticodon loop of ordinary t-RNA from U31 to A39 (U-OMC-U-C-C-A-MIA-A-A), the anticodon of which is for Gly. It is, however, important to notice that polymerization of L-amino acids seems far more favorable than that of D-amino acids. Only one D-amino acid in a consecutive L-amino acid peptide could be involved, but does not seem favorable. Such a predilection for L-amino acids becomes much clearer in the ancestral form of amino acylation as shown in the following stereo pictures showing from glycine (Gly) in Figure 9 to tryptophan (Trp) in Figure 31. In these pictures O4 atom of U33 is not hydrogen-bonded to the phosphate oxygen of 35th position that is always the case for all crystallographic results of tRNAs [8]. It is known that U33 is highly conserved for all tRNAs. The anticodon loop holds in its crystal structure a magnesium ion in the pocket, but under a low magnesium concentration of 1 mMol, that is about the same as that of outside the cell, the loop behaves much more flexible than that of crystalline form [9]. This is one of the most important point in the evolution of life for capability of selection for all L-amino acids and glycine in its pocket. At this stage of evolution of life. The nucleotide at the 33th position might not have to be uridine, but U33 seems more favorable for performing a multifunctional role in translation [9].
At the periphery of the polyuridine layers condensation of amino acids with its carboxyl end to 3’-end of a terminal uridine promotes polymerization of small amino acids like glycine, alanine, serine, valine, threonine, and proline. The anticodon loop structure seems to have evolved spontaneously so that it could catch only L-amino acids. It can be seen that D-amino acids were very hard to be accommodated in the cavity of the anticodon loop. However, how the respective nucleotide sequence of the anticodons were favorable for the nominated amino acid by the chemical structure of the codons, such as, N6 of A, O6 and N2 of G, N4 of C, and O4 of U. Accordingly, there could be a lot of mistakes in selecting amino acid by the nucleotide combination of anticodon. In order to enhance the accuracy of amino acid selection by the combination of anticodon, we must wait a long time before evolution of aminoacyl tRNA synthethase as well as tRNA itself. As is well known, the length of tRNA has become evolved to about 76 nucleotides of clover-leaf shape. The total three-dimensional structure of tRNAs are referred to as the L-shaped form, the waist region of which is particularly important for amino acid type recognition. On the other hand, the evolution of aminoacyl tRNA synthethase seemed to have started to make a dimer of a couple of synthetase and tRNA. Because of the elongation of the distance between the 3’-end and the anticodon loop of a tRNA during the course of evolution, the 1 to 1 recognition of anticodon with its 3’-end of tRNA became more and more difficult, so that a pair-wise recognition of anticodon loop with its 3’ end of the partner tRNA might have been more easier to recognize. The evolution of aminoacyl-tRNA synthetase (aaRS) has led to two classes, Class I and Class II, [10,11] based on structural sequence features of the catalytic core. Each aaRS recognizes a single amino acid and attaches it to the 3’-end of a tRNA whose anticodon matches a codon for that amino acid. According to Ibba and Sӧll [12], however, there exists many exceptions to these rules, and many aaRS additionally rely on various proofreading or editing mechanisms to ensure the translation to the genetic code. In order to charge a tRNA, the amino acid must be first activated with ATP by the aaRS to form an aminoacyl-adenylate intermediate and then transferred onto the terminal adenosine of the tRNA [12].

After aminoacylation, two tRNAs are able to be arranged by a short sequence of nucleotides so that a dipeptide of Pro-Gly may be preferable for supporting the total structure of the system such as a part of aminoacyl tRNA synthetase. Figure 32 shows the simplest case of protein synthesis showing an ancestral form of mRNA and synthesized protein Pro-Gly. Accuracy of protein synthesis in accordance with the prescription of mRNA could be best for Gly and Pro at the first stage of evolution of life. As accuracy of aminoacylation is enhanced, a variety of peptide becomes ready for preparation by use of a special set of mRNA prepared for a necessity of growth of nucleotide layer. Since the number of L-amino acids was increasing, the cavity of the 6-fold symmetrical layer became a little too narrow, so that the next stage of more compact and a little wider in cavity might have been useful. Figures 33 and 34 show two stereo views of 6.5 fold symmetrical left-handed single-stranded polyuridine. In this case, there is no need of worrying about the short contact between the nucleotides of two nearest neighbors, and the stacking base distance is also all right. However, a lot of pyrophosphonucleotides and phospholipids are excluded. Figure 35 shows that a peptide composed of L-amino acids having rather small side chains such as Ala, Ser, Val, Pro and Gly can be accommodated inside the cavity of the left-handed helical cylinder, but those having large side chains could have a tendency to break the regularity. Arg residues in particular tends to disrupt the regular shape, particularly because the helical cylinder has a consecutive line of phosphate groups inside, resulting in a familiar shape of right-handed double-stranded polyuridine helices. Since such a role of Arg residues were so important, Arg occupies a comparably large part in the Table of the genetic code in comparison with that of Ser, if we are allowed to think that Gly, Ala, and Pro were comparatively important to the other amino acids in the earlier stage of evolution of L-amino acid proteins. Similarly, Leu and Val residues seem to have been more important in playing a role of making hydrophobic side of α–helices and β–sheets. The roles of Glu, Asp, His and Lys could have been evolving as players of catalytic activities. It is well known that some particular form of RNAs could have a catalytic activity [13-15]. However, there seems to be a handicap of flexibility in the 3D-structure of RNA, so that the evolution of proteins having Glu, Asp, His and Lys residues became more and more important during the course of the origin of life. Also, important to consider the double-stranded right-handed RNA helices, it is immediately related with the importance of the canonical G-C and A-U base pairs and G-U wobble base pair. The growing of double-stranded regions of RNA made it possible of the evolutions of tRNA, mRNA and ribosomes of both large and small subunits.

Evolution of ribosome

The significance of the ribosome in the origins of life consists in the movement of mRNA between two subunits of the large and small RNA fragments. At the beginning of the origin of life, when no protein factors such as elongation factors Tu and G as well as rRNA binding proteins existed, there was no energetical barriers between A and P sites, and also between P and E sites. Brownian motions of the conformational changes performed the translocation of the ancestral forms of tRNAs accompanying splitting and uniting of RNA fragments. On the other hand, the structures of the present-day ribosome are too big. Figure 36 shows the secondary structure of Esherichia coli 16S rRNA, which consists of 1542 nucleotides and 21 binding proteins, and Figures 37 and 38 show 5’-half and 3’-half of the secondary structures of E. coli 23S rRNA, which consist 2904 nucleotides and 30 binding proteins. There are 5 different families in the living organisms in the present world. Those are (a) Eubacteria, (b) Archaea, (c) Chloroplast, (d) Eukarya, and (e) Mitochondria. The secondary structures of 52 organisms in (a), 25 organisms in (b), 27 organisms in (c), and 44 organisms in (d) were analyzed, and the references of those sequences were listed in Nagano and Nagano [7]. The analyses have shown that the simplest secondary structures of the ribosome are mitochondrial rRNAs of Trypanosoma brucei, 9S small subunit rRNA of which is shown in Figure 39, and 12S large subunit rRNA of which is shown in Figure 40 [7]. It is said that the smallest rRNA of mitochondrial protista Trypanosoma brucei is stabilized by binding comparatively more proteins. However, at the period of the ancestral ribosome, evolution of binding proteins is supposed to be immature, and so, the ribosome was supposed to stick to the silicate rocks at the bottom of the volcanic hot spring, where the concentration of alkaline metals including Mg2+ ions are relatively low [4], and temperature and pressure were very high. The Brownian motion of the ancestral ribosome were suppressed by the high pressure instead of binding proteins.

It has been known that the first step of codon-anticodon recognition only expels the ternary complex of noncognate aa-tRNAs with accuracy of about 200/10,000 [16]. The mechanism of kinetic proofreading at the step of GTP hydrolysis was proposed to expel the ternary complex of near-cognate aa-tRNA to enhance the accuracy up to 5/10,000 [17]. Here, the ternary complex denotes a complex of aa-tRNA, elongation factor Tu (EF Tu), and GTP. It was found, however, the accuracy is reduced by the used of high Mg2+ concentration [18]. Thompson, et al. [19] has observed that incorporation of near-cognate aa-tRNA into A site sharply increases from 6 mMol of Mg2+ concentration and reaches a plateau at 12 mMol Mg2+. The present author tries to explain how such a phenomenon could be realized on the 3D structure of the ribosome.

The 12 nucleotide sequence of 23S rRNA, AGUACGAGAGGA, is the longest universally conserved among all known rRNAs [20]. The present author has found that the region of nucleotides 1064-1077 of 16S rRNA in E. coli sequence is complementary with the region of nucleotides 2653-2666 of 23S rRNA, that is called sarcin-ricin loop (SRL) [7]. This relationship holds for all nucleotide sequences listed in the Figure 41 for Eubacteria, Figure 42 for Archaea, Figure 43 for Chloroplast, Figure 44 for Eukarya, and Figure 45 for Mitochondria. Since the region 2653-2666 of 23S rRNA is called GTPase binding site, and the nucleotides at the positions 1068-1073 of 16S rRNA constitute the 5’-side of secondary structure, helix 35 of 16S rRNA (h35), and those of 2653-2658 and 2663-2666 of 23S rRNA are the head region of helix 95 (H95), the relationship can be summarized as shown in Figure 46, in which SpcF denotes spectinomycin footprint site [21]. The residue number 2660 indicates the sequence of α–sarcin cleavage site [22]. The left-most structure in Figure 46 plays an important role in GTP hydrolysis, while another structure on the second from the rightmost side seemed to be that of transition state, but finally turned out to be the binding site of GTPase at the moment of GTP hydrolysis. Although such a long helix has been observed by neither X-ray analysis nor cryo-EM, a model building technique can show how it looks like, as shown in Figure47. The reason why some bases are unpaired in this model is that the corresponding nucleotides in Eukarya, as shown in Figure 44, are G-G unfavorable pairs.

U33-extended and U33-folded forms of the anticodon loop of tRNA

Uridine 33 at the 5’-side of the anticodon loop of tRNA is universally conserved except for cytidine 33 in some initiator formylmethionyl-tRNAs. The uridine base of U33 is folded inside the anticodon loop of tRNAs observed so far by X-ray crystallography, and hydrogen-bonded to a phosphate oxygen atom of 36th nucleotide of the anticodon [8]. This structure is referred to as U33-folded form in this article. This conformation of the codon-anticodon heix of U33-folded form does not seem stable enough. The U33 base can be expected to stick out or to be exposed if the conformation of the codon-anticodon helix assumes that of an ideal A-form RNA. Such a structure could be a little bit unstable because of the exposed U33 base compared to the hydrogen-bonded U33 base of the U33-folded form. And yet, the stability of the U33-extended form heavily depends on the stability of the codon-anticodon helix, and seems to play an important role in enhancing the accuracy of translation. The relative stability between the U33-folded and extended forms must also depend on the concentration of Mg2+ of the buffer solution used in the experiments. It is said that the Mg2+ concentration inside muscle cells is about 1 mMol that is about the same as that outside the cells. On the other hand, a Mg2+ concentration used for crystallization of whole ribosome is 10 mMol or much higher in some cases. The conformation of the anticodon loop of tRNAs in the living cell must be more dynamic. The difference in the positively charged ions between the living ribosomes and the ribosome crystals are mainly compensated by K+, if the environment of ribosomes are about the same as the muscle cells. A recent result of X-ray structure of the decoding site has shown that the G-U base pairs of near-cognate codon-anticodon interactions at the first position (between mRNA/Phe and tRNA2Leu) and at the second position (between mRNA/Cys and tRNATyr) cannot be discriminated from the cognate G-C and A-U base pairs, respectively [23]. The sharp increase of the near-cognate GTPase reaction between poly(U)-programmed ribosomes and Leu2 ternary complexes from 6 mMol to 12 mMol (Figure 1 [19]) strongly suggests that deprotonation of N3 and protonation of O4 of uridine base of G-U pair cannot be distinguished from G-C pair [23], and that the conformational change of the anticodon loops from the U33-extended to U33-folded form of the ribosomes do not discriminate the near-cognate Leu2-tRNA from the cognate Phe-tRNA. Figure 48 shows a different 3D structure for the near-cognate G-U pair at the first position of the codon-anticodon helix on the basis of the model building technique of the present work, in which the U33 base, which is drawn in magenta, sticks out from the anticodon loop, although a Mg2+ hexahydrate still binds between OP2 atom of YG37 and O2 atom of O2’ methylated OMC32. The U33-extended form of the anticodon loop would destabilize the loop structure. Besides, deprotonation from N1-H of guanine base and N3-H of uridine base resulting in the movement of the proton to the O4 atom of the uridine base might be preferable under the Mg2+ excess conditions. Figure 1 of Demeshkina, et al. [23] also shows that P/A-kink of the mRNA is widely open as the kinks obtained by the other X-ray structures of the ribosomes [24,25] and supported by binding two clusters of Mg2+ hexahydrate. On the other hand, Nagano (2015) demonstrated a different type of P/A- and E/P-kinks, as shown in Figure 49, each of which is supported by one cluster of Mg2+ hexahydrate. The difference of the kink structures of Nagano [9] from those in the X-ray structures can be rationalized on the basis of the low Mg2+ concentration described above. In contrast to Figure 48, Figures 50 and 51 show the structural difference in the A-site codon-anticodon helix between the cognate case in Figure 50 and the near-cognate case in Figure 51. In these two structures, the bases of both U33 and OMC32 as well as YG37 are exposed in order to represent a delicate difference between the cognate and near-cognate codon-anticodon helices. As a result of the difference, a Mg2+ hexahydrate between OP2 atom of YG37 and O2 atom of OMC32 is expelled, giving rise to a slight difference in the interactions between the bases of U33 and A1396. The interactions of A1492 and A1493 to the minor groove of the codon-anticodon helix can be achieved almost the same as those in the X-ray structures [23], but the interaction of G530 is rather difficult without a conformational change in the 530 loop due to the big conformational change in the anticodon stem-loop (ASL) structure as well as the big shift in the location of h45, which will be described later. The near-cognate G-U base pair at the first bases of the A-site codon in Figure 51 is quite the same as that of Figure 48, but resulting in a delicate but recognizable difference from the cognate case in Figure 50, while the structure shown in Figure 48 cannot be achieved because of its big structural difference from the corresponding one in Demeshkina, et al. [23].

The exposed base of U33 in the U33-extended form is supposed to be supported by base-pairing with a certain universally conserved adenine base at the decoding site of 16S rRNA, the secondary structure of which is shown in Figure 36. Such base pairs are long-range interactions in contrast to the hydrogen-bonded U33 base inside the anticodon loop so far observed in X-ray structures. Accordingly, a transition between U33-folded and U33-extended forms would be more frequent in the physiological condition of the Mg2+ concentration. Although the G-C pair between C1399 and G1504 has been predicted [14,15] because of its high conservation, it was not confirmed in the crystal structure of 2HGR (accession number in PDB database [26]). On the other hand, G1401-C1501 pair as well as the base stacking of C1400 to the first base of the anticodon of P-site tRNA [27] was confirmed. It has been known that both adenines and cytidines in the decoding site are universally conserved. Accordingly, it was quite natural to think that U33 base of the U33-extended form of P-site tRNA could be base-paired with A1398. It necessarily leads to the assignment of A1396 to the base-pairing partner of the U33-extended form of aa-tRNA at the recognition mode of A site (T site). It seemed possible for either A1502 or A1503 with U33 of the U33-extended form of deacylated tRNA at the E site. The result of the model building of Nagano (2015) has shown that A1502 is most favorable, while A1503 seems a little too far from the U33 of E-site tRNA.

A-P and P-E tRNA docking pairs in the pre- and post-translocational states

The universal rule of nucleotide sequence complementarities (Figure 46) was first noticed by a finding that oligonucleotide fragments such as TѰCG or UUCG inhibited the binding of aa-tRNA at the A site [28]. It was found that such a highly conserved sequence exists on the 3’-side of h35 of 16S rRNA, and that the other side of h35 is complementary to the conserved loophead sequence of SRL in 23S rRNA [29]. In addition to the above finding, it was also found that various tRNA fragments containing D-loop inhibited aa-tRNA binding at the A site [28]. This strongly suggested that the T-loop of aa-tRNA making base-pairs with the 3’-side of h35 and the D-loop of aa-tRNA making base-pairs with the T-loop of P-site tRNA are an essential necessary requirement for the aa-tRNA entering the A site. It is also important to note that there exists a pair of highly conserved guanines at the almost all tRNAs (although their relative locations are slightly different from one another), while that the highly-conserved cytidine residues in T-loop of tRNAs are C56 and C61. (However, there exists an exceptional case in human mitochondrial Ser-tRNA missing totally its D-loop. How we can interpret its translational movement? That is an important question.) In contrast to the X-ray structure of the elbow region of the isolated L-form tRNA, in which G19 is paired with C56 as a Watson-Crick type, but G18 is hydrogen-bonded to Ѱ55 in an irregular fashion [8], the universally conserved guanines, G18 and G19, of aa-tRNA at the T site are base-paired with the other universally conserved cytidines, C56 and C61, of P-site tRNA, respectively, and also G18 and G19 of P-site tRNA with C56 and C61 of E-site tRNA, respectively. Such a big difference in the shape of tRNA between the isolated L-form of X-ray structure [8,21,26] and the docking model of Nagano et al. [29] as well as Nagano [9] comes from a big difference in the Mg2+ concentration, as mentioned above.

The results of X-ray structures of the whole ribosome have shown that two conserved regions of 16S rRNA, the ridge containing G1338 and A1339 near h29 and the head of 790 loop, constitute barriers that block a translocational movement of P-site bound tRNA toward E site, if we assume that such a movement is just a simple-minded translation from the crystallographic P site to the crystallographic E site. Transition from the X-ray structure to the predicted one of Nagano [9] would start from the U33-extended form of aa-tRNA by interaction between U33 (aa-tRNA) and A1396, which is exposed on the X-ray structure, as well as the base-pairing of GTѰCG (or GTѰCA) of aa-tRNA with CGAGC1107. The effector region in the body of elongation factors, both EF-Tu and EG-G, would collide with the long helix composed of h30, h32, and h34 [14,15], resulting in accomplishment of the long-range interactions between the SRL-region of 23S rRNA and the 5’-side of h35 of 16S rRNA. Under the condition of fixing the location of A76 of both P- and E-site tRNAs, the U33-extended forms make the distance between G18 (P-tRNA) and C56 (E-tRNA) shorter and C56 (P-tRNA) closer to G18 (aa-tRNA), and, the twisting makes splitting ofD-loop from T-loop for both P- and E-site tRNAs at their respective elbow regions. Such a conformational change for both P- and E-site tRNAs allows base-pairing between G18 (aa-tRNA) and C56 (P-tRNA) and between G18 (P-tRNA) and C56 (E-tRNA), and successively, G-C pairs between G19 (aa-tRNA) and C61 (P-tRNA) and between G19 (P-tRNA) and C61 (E-tRNA), respectively. Thus, the A-P-E tRNA docking triplet form can be completed, and finally accomplishes the codon-anticodon helix at the E site. This is a series of conformational changes after unfolding of h35 by the collision with the body of elongation factors, EF-Tu and EF-G, ending up with formation of the codon-anticodon helix at the E site.

Figure 52 shows that U33 at the P site (green) is base-paired with A1398 (red), U33 at the E site (purple) is base-paired with A1502 (pink), but U33 at the A site (magenta) is not base-paired with A1396 (brown). C1399-G1504 pair is formed and shown in gold. Figure 53 shows that U33 at the P site (green) is base-paired with A1398 (red), U33 at the A site (magenta) base-paired with A1396 (brown), but U33 at the E site (purple) is not base-paired with A1502 (pink). C1399-G1504 pair (gold) is formed. On the other hand, Figure 54 shows that all three U33 at the A, P and E sites, (magenta, green, and purple, respectively), are base-paired with A1396 (brown), A1398 (red), and A1502 (pink), respectively, at the expense of separation of the bases of C1399 and G1504 (gold). Since both tRNAs at the P and E sites are cognate, in contrast to the case of X-ray structure, aa-tRNA at the T site would take a transition-state conformation, as shown in Figure 54, if it is also cognate. During fluctuation of the whole structure, both residues, C1399 and G1504, would become close enough to base-pair, as they are not so far separated. Then, A-U base pair of either A site or E site is broken. Which A-U pair is broken depends on the stability of the codon-anticodon helices at the A and E sites, since the distance between the U33 base and C1400 base, for example, would become larger in the case of less stable anticodon loop. From this reason, both noncognate (before GTP hydrolysis) and near-cognate aa-tRNA (at the step of GTP hydrolysis) are rejected, while only cognate one (after GTP hydrolysis) can enter the A site to expel the deacylated tRNA at the E site from the ribosome. This would be a reasonable explanation of the mechanism of translation concerning how high accuracy could be achieved at the decoding site.

When the region of U2653-C2666 of 23S rRNA (magenta) including the SRL is bound together with the region of G1064-G1077 of 16S rRNA (skyblue), the conformation of which is shown in Figure 47, the region of 16S rRNA is extended from the main part of 30S subunit. In order to achieve such a long-range base-pairing interactions, it is essential that the long helix composed of h30, h32 and h34 of 16S rRNA should have bends of a considerable degree around the regions between h32 and h34 as well as between h30 and h32. It necessarily causes a big deviation of the barrier, G1338 and A1339, toward the joint region of h32 and h34 from its original position, and the location of h45 is changed very much. As a result, the universally conserved dimethylated adenines, Am21519 and Am21520, at the loop of h45 makes a contact with the downstream of mRNA. This result can explain why those residues are dimethylated, while the X-ray structure, 2HGR [26] cannot. A1396 approaches the U33 base of aa-tRNA, while h44 of 16S rRNA makes a contact at the inner groove of A-site codon, with universally conserved A1492 and A1493 [30]. When U33 of the A-site tRNA is base-paired with A1396 and U33 of the E-site tRNA is not base-paired with A1502, as shown in Figure 53, the deacylated tRNA at the E site (purple) in the three tRNA binding structure changes its conformation from the U33-extended and docking with P-site tRNA state to the U33-folded and isolated form. Then, the deacylated tRNA leaves the ribosome in the direction of L1 stalk. When the conformations of nucleotides at the decoding site are such as those in Figure 52, what would happen to the T site? When the aa-tRNA is noncognate, the base-pair of the codon-anticodon helix would not be formed without need of U33-A1396 base-pair formation. In the case of either near-cognate or cognate, the codon-anticodon helix could become an ideal A-form, and the help of U33-A1396 would be very important at the step of GTP hydrolysis. After fluctuation of the conformations at the decoding site between those as in Figures 54 and 52, the helix at the T site would be broken, if the one at the E site is a little stronger. Then, the conformation of the aa-tRNA would become a U33-folded form with its isolated L-form elbow region, and leaves the ribosome in the direction of L7/L12 stalk. At the instant, the conformation of the A-site codon would loosen, and become ready for accepting the next anticodon of aa-tRNA as a ternary complex, that would have probably been waiting for opening the A-site codon by using the 3’-side of h35 and A1394. A1394 is also highly conserved, although it is less conserved than A1396 and A1398.

Translocation as a rotational movement of A-P tRNA docking pair to P-E pair

The inhibition of spectinomycin on the EF-G-dependent translocation has been noticed long time ago [31,32]. In addition to the above, Peske et al. [33] have concluded that EF-G produces conformational rearrangements on the ribosome in the vicinity of the decoding region on the 30S subunit and that spectinomycin has a direct inhibitory effect on tRNA-mRNA movement, that is quite consistent with the basic assumptions of the present work. Comparison of Figure 56 with Figures 57 and 58 suggests that translocation is not a translational movement of tRNAs from the crystallographically obtained P site to the E site but a rotational one around an axis that is close to both A- and E-site tRNAs. If the aminoacyl group remains at the A site and the P-site tRNA is still peptidylated, translocation would not occur because of locking at the P site [34]. Peptidyl transferase reaction results in peptidyl-tRNA at the A site and deacylated tRNA at the P site, the state of which automatically brings about P and E sites on the 50S subunit. This is due to a rotational freedom around the hinge between H14 and H22 (nucleotides AA423) and a rigid binding of two helices, H22 and H88 at a pseudoknot CCAU416/AUGG2410 of 23S rRNA. C2394 and G2421 at the base of H88, which is empty as a result of the process shown in Figure 57, could turn to catch the adenine base of the CCA end of deacylated tRNA at the P site [35]. According to chemical modification experiments, 2’-hydroxyl groups of the nucleotides 71 and 76 of the deacylated P-site tRNA can be recognized by nucleotide C1892 in the minor groove of H68 and A2433 and A2434 at the base of H74 of 23S rRNA, respectively [36]. Those residues also constitute part of the E site on the 50S subunit. It would expel the P-loop at the loop of H80 as well as A2450 and its associated nucleotides of the central loop of 50S subunit from the CCA end of P-site tRNA, that is the P site on the 50S subunit, and transfer them to the CCA end of the neighboring A-site tRNA. These are what we call P/E and A/P hybrid states. Such a transition would not occur if either the 3’-end of P-site tRNA is peptidylated or some of the above key nucleotides are chemically modified. This could be an explanation as to why peptidyl-tRNA bound to the P site locks ribosomal functions such as translocation, termination, and recycling [34]. Figure 59 shows that the foot of H88 (cyan), where C2395 and C2421 are situated, reaches the adenine base of P-site tRNA (green), while the P-loop of H80 (red) catches the bases of C74 and C75 of A-site tRNA (magenta). This is the 3D structural representation of the 3’-end of tRNAs at the A/P and P/E hybrid states. When EF-G·GTP complex comes to occupy the space of A-site tRNA, the A-P tRNA docking pair rotates around the axis passing closer to the A-, P- and E-site tRNAs to reach the site of P-E tRNA docking pair at the classical P/P and E/E states. The 3D structural representation of them can be shown as in Figure 60.

How elongation factors work on the present models to produce GTP hydrolysis?

The recent result of the crystal structure of the ribosome bound to EF-Tu and aa-tRNA [37] does not seem to fully explain the mechanism of accommodation of aa-tRNA into A site. Starting from the tRNA structure in magenta in Figure 58, which is either leaving the ribosome or entering the ribosome as a ternary complex with EF-Tu·GTP, its 3’-end region was modified by transplanting 3’-end region of Trp-tRNATrp along with EF-Tu and GDP bound to a cognate codon on the 70S ribosome 2Y18 [38]. GDP structure was changed to GTP by adding Pγ phosphate. The conformation of EF-Tu was changed so that the Pγ atom comes closer to N6 atom of A2660 and Pβ atom closer to N4 atom of C2658. Pγ and Pβ atoms are covered by the region of amino acid residues 20-26 (VDHGKTT) in EF-Tu. The G base of GTP moiety is hydrogen bonded to U2656, while the O3’ and O2’ atoms on its ribose ring are hydrogen-bonded to N6 atom of A2657 and O6 atom of G1074 of 16S rRNA, respectively. Such a local structure of GTP binding to the present model at the interface of RSL of 23S rRNA and the 5’-half of h35 of 16S rRNA on the ribosome structure is shown in Figure 61 [9].

Inspection of this local structure suggests that GTP hydrolysis could occur when the region of EF-Tu with GTP moves in the direction from N4 atom of C2658 to N6 atom of A2660 in contrast to the movement vertical to the above. When the distance between the N4 and N6 atoms becomes larger accompanying the Pβ and Pγ atoms, respectively, NH2 group of the N2 position of G2659 attacks the Pβ-Pγ bond. The total structure of EF-Tu with the related regions of T- and P-site tRNAs is shown in Figure 62. After GTP hydrolysis in the structure shown in Figure 62, the conformation of EF-Tu returns to the original one observed in the crystal structure of the ribosome bound to EF-Tu and Trp-tRNATrp 2Y18 [38]. Such a structure is shown in Figure 63. The EF-Tu molecule would leave the ribosome soon under the normal condition of low Mg2+ concentration. The conformation of the A-site tRNA is the same as those shown in Figures 56 and 57. Since the binding of A-site tRNA very close to P-site tRNA, the location of which is almost the same as the other structures shown in Figures 55 to 58, the cavity between the SRL region and P-site tRNA becomes a little wider, where EF-G is supposed to bind. The structure of tRNA in gold, as shown in Figure 64, is a putative ancestral form of EF-G, in which the tRNA-like body in gold represents the domain IV of EF-G [39], while the other part represents the rest of the whole structure of EF-Tu. If the conformational change in the part of EF-Tu occurs after GTP hydrolysis, the structure very much like the one shown in Figure 62 would be obtained. Because the exposed form of the domain IV of EF-G, or the tRNA moiety in gold, would be very unstable, the structure would become immediately the same as that in Figure 63 after translocation. This is exactly what happened in the crystal structure of the ribosome shortly after binding of EF-G [40]. The reason why EF-G dependent translocation is inhibited by binding of spectinomycin [31-33] can be explained very clearly as the result of missing the contribution from the 5’-half of h35 in the mechanism of GTP hydrolysis shown in Figure 61. In contrast to the EF-G dependent translocation, the A-site tRNA accommodation catalyzed by EF-Tu does not seem to be affected by spectinomycin. The reason for this would be that the region of CGAGC1107 on the 3’-half of h35 is unfolded by interaction with the GTѰCG region of T-loop of aa-tRNA, or could be due to a slight difference in the distance between Pγatom and 3’-end of P-site tRNA (27 Å). GTP hydrolysis results in a big conformational change in EF-Tu with a right hand (amino acid sequence from Ala1 to Arg190) pushing the GTPase-binding site and with a left hand (from Gly191 to Glu405) pulling the whole body of P-site tRNA. The final shape of A-site tRNA is just like a sitting-up of the T-site tRNA. On the other hand, GTP hydrolysis in translocation by means of EF-G produces a little different result from that of EF-Tu. This could be due to a slight difference in amino acid sequence between EF-Tu and EF-G (one amino acid insertion) and result in a different rotational movement with EF-G’s left hand pushing the whole body of A-site tRNA toward P-site tRNA and with its right hand pushing the GTPase-binding site to break the long helix made by the SRL region of 23S rRNA and 5’-side of h35 of 16S rRNA. The big distortion introduced by formation of the GTPase-binding site is cancelled and returns to the original state. This movement of the whole ribosome represents exactly where the driving force of the ratchet-like movement of the ribosome [41] comes from.

Evolution of ribosomal binding proteins and protein factors

During the course of evolution of ribosome, various ribosomal binding proteins have also pursued their structural evolutions by changing their amino acid sequences. The present author has been carrying a question as to what type of amino acid sequence can selectively recognize their responsible locations on the 16S and 23S rRNAs. The number of ribosomal proteins is found to range between 50 – 60 in bacterial ribosomes and 70 – 80 in cytoplasmic ribosomes of eukaryotes [42]. In E. coli there are 21 small-subunit ribosomal proteins and 32 individual large-subunit ribosomal proteins [42]. Using large subunit ribosomal proteins and ribosome (50S) from E. coli, Nierhaus performed the analysis on the stepwise assembly of 50S ribosome by reconstitution [43]. Nierhaus’s 50S assembly diagram shows that large subunit ribosomal proteins, L2, L3, and L4, recognizes somewhere on the 23S rRNA sequence around 1500, 2200 and 650, respectively. Figure 65 shows that L2 from E. coli has a considerable sequence homology with those from Paenibacillus pabuli (P. pab) and Thermus thermophillus (T. ther), L3 from E. coli with those from Alicyclobacillus sendaiensis (A. sen) and T. ther, and L4 from E. coli with those from Bacillus vallisimortis (B. vall) and T. ther. The three-dimensional atomic models of the present article are based on the Cartesian coordinates of Korosterev et al. [26] (PDB accession numbers are 2HGR for small subunit and 2HGU for large subunit). The locations of all ribosomal proteins for both 16S and 23S subunits are shown on the atomic models of the present work as from Figures 66 to 111. If we compare the sequence comparison of L2, L3 and L4, as shown in Figure 65 with the atomic coordinates of 23S rRNA and those of L2, L3 and L4, we might be able to obtain something about the idea about the strength of the interactions between peptide structure and rRNA structure. In that case the result of the analysis of step-wise reconstitution of 50S subunit [43] will give us a lot of information about the protein-rRNA and protein-protein interactions. Such an analysis has not been done yet.

Figure 112 shows the similarity of amino acid sequences of the effector region for four types of GTPases, viz. EF-Tu and its related elongation factors (eEF-1), EF-G and its related ones (eEF-2), termination factors (RF3), and initiation factors (IF-2). The proteins in the group of EF-Tu has a common sequence type such as D****E**RGITI, while those in the group of EF-G has a sequence type of D****E***G**I, and those in the group of RF3 has a sequence type of D****E**RGIS*. It seems important to note that all of them except the group of IF-2 started their evolution from a common sequence of GTPase, but gradually diverged into three different sequences for performing a slightly different function with respect to 70S ribosome. It is, however, a quite different function with initiation factors that must start their function by splitting the 70S ribosome into 30S and 50S subunits. The amino acid sequence of the effector region of IF-2 seems to tell us that it took a long time to reach the present state if it also started its evolution from the common structure of the ancestral GTPase. The personal opinion of the present author is that the RNA structure of the ribosome would have started at the bottom of the mantle under the strong influence of silicate rocks without much help of protein factors and binding ribosomal proteins, when their environment, in particular, was controlled by high pressure and temperature, and that the evolution of various types of protein structures helped the ancestral form of ribosomes to be independent of the silicate rocks. There must have been an increasing trend of water concentration as well as alkali metal concentration. In order to protect the hydrolytic effect of such trends, evolution of ribosomal proteins and cell membrane structure must have been very important. This is the evolution of ribosome, that is also highly associated with the evolution of aminoacyl-tRNA synthetase, very briefly described above. The story about the origin of life begins from the completion of ribosome structure and how an assembly of messenger RNAs performs self-reproduction before appearance of DNA in the world. This is the story of RNA world [6].

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Editorial Information

Editor-in-Chief

Ivan Gout
University College London
Ricardo H. Alvarez
Cancer Treatment Centers of America

Article Type

Review Article

Publication history

Received: April 10, 2017
Accepted: April 21, 2017
Published: April 25, 2017

Copyright

© 2017 Nagano K. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Nagano K (2017) Abiogenic evolution of ribonucleic acid as a left-handed helix and its protecting substances on primordial earth – A story about evolution of ribosome and the origin of life. Integr Mol Med 4: DOI: 10.15761/IMM.1000284

Corresponding author

Kozo Nagano

Nagano Research Institute of Molecular Biology, 4-8-24 Higiriyama, Kohnan-ku, Yokohama 233-0015, Japan

Figure 1. Stereoview of 6-fold left-handed single-stranded helix of 5’-phospho-D-ribose.

Figure 2. Stereoview of three 6-fold left-handed single-stranded helices of 5’-phospho- D-ribose hydrogen bonded by U-U base pairs, which are shown in a different direction from that of Figure 1.

Figure 3. Stereoview of the contact area of the three infinite helices intercalating one molecule of adenine triphosphate (ATP) which is drawn in green.

Figure 4. Stereoview of polymerized peptides, amino acid sequence of which are Gly-Pro- Gly-Asp-Ala-Ala-Ser-Val. The conformation of the peptides in blue is not α–helix.

Figure 5. Stereoview of dimerization of glycine and proline. Glycine drawn in pink is bonded to 3’-end of polyuridine at its bottom region of the left-handed single-stranded helix. N-terminal end of proline in blue is bonded to carboxyl end of glycine.

Figure 6. Stereoview of dimerization of tryptophan and proline. Tryptophan drawn in pink is bonded to 3’-end of polyuridine at its bottom region of the left-handed single-stranded helix. N-terminal end of prolin in blue is bonded to carboxyl end of tryptophan. This picture is almost the same as that of Figure 5 except the side chain of tryptophan which is the largest among all L-amino acids.

Figure 7. Stereoview showing a dipeptide Gly-Pro is bound at 3’-O of polyuridine terminus on the long helix in brown which is separated from the other part of long helix (just one turn) in yellow. The nucleotides in magenta is a part of anticodon loop of ordinary tRNA from U31 to A39 (U-OMC-U-C-C-A-MIA-A-A), the anticodon of which is for Gly. The rest of polyuridine in helix is drawn in orange for P atom, red for O, and white for C.

Figure 8. This picture corresponds to Figure 7 showing the anticodon in violet. Besides, ATP molecule is drawn in gold. The reason why ATP sits in contact with 3’-O of uridine and carboxyl end of Gly is that an extra amount of energy is needed for aminoacylation during the course of lowering temperature and pressure of the environment.

Figure 9. This picture shows an ancestral form of aminoacylation for Gly. The other parts are the same as the preceding picture, except for Cα in orange, Ccarboxyl in blue, and two carboxyl oxygens in red. This picture is for that of transition state before one of the carboxyl oxygen is removed by dephosphorylation of ATP.

Figure 10. This picture is the same as Figure 9 except for Ala. Cα and Cβ are drawn in orange. Anticodon nucleotides in skyblue are CGC.

Figure 11. This picture is the same as Figure 9 except for Pro. Cα, Cβ, Cγ and Cδ are drawn in orange. Anticodon nucleotides in skyblue are CGG.

Figure 12. This picture is the same as Figure 9 except for Ser. Cα and Cβ are drawn in orange, and Oγ is in red. Anticodon nucleotides in skyblue are GCU.

Figure 13. This picture is the same as Figure 9 except for Arg. Cα, Cβ, Cγ, Cδ, and Cζ are drawn in orange, and Nε, NH1 and NH2 are in blue. Anticodon nucleotides in skyblue are CCU.

Figure 14. This picture is the same as Figure 13 for Arg except for anticodon. Cα, Cβ, Cγ, Cδ, and Cζ are drawn in orange, and Nε, NH1 and NH2 are in blue. Anticodon nucleotides in skyblue are CCG.

Figure 15. This picture is the same as Figure 9 except for Val. Cα, Cβ, Cγ1 and Cγ2 are drawn in orange. Anticodon nucleotides in skyblue are CAC.

Figure 16. This picture is the same as Figure 9 except for Thr. Cα, Cβ and Cγ1 are drawn in orange, and Oγ2 is in red. Anticodon nucleotides in skyblue are CGU.

Figure 17. This picture is the same as Figure 9 except for Ser. Cα and Cβ are drawn in orange, and Oγ is in red. Anticodon nucleotides in skyblue are CGA.

Figure 18. This picture is the same as Figure 9 except for Met. Cα, Cβ, Cγ and Cε are drawn in orange, and Sδ is in green. Anticodon nucleotides in skyblue are CAU.

Figure 19. This picture is the same as Figure 9 except for Ile. Cα, Cβ, Cγ1, Cδ and Cγ2 are drawn in orange. Anticodon nucleotides in skyblue are GAU.

Figure 20. This picture is the same as Figure 9 except for Leu. Cα, Cβ, Cγ, Cδ1 and Cδ2 are drawn in orange. Anticodon nucleotides in skyblue are CAG.

Figure 21. This picture is the same as Figure 20 for Leu except for Anticodon. Cα, Cβ, Cγ, Cδ1 and Cδ2 are drawn in orange. Anticodon nucleotides in skyblue are CAA.

Figure 22. This picture is the same as Figure 9 except for Glu. Cα, Cβ, Cγ and Cδ are drawn in orange, and Oε1 and Oε2 are in red. Anticodon nucleotides in skyblue are CUC.

Figure 23. This picture is the same as Figure 9 except for Asp. Cα, Cβ and Cγ are drawn in orange, and Oδ1 and Oδ2 are in red. Anticodon nucleotides in skyblue are GUC.

Figure 24. This picture is the same as Figure 9 except for Gln. Cα, Cβ, Cγ and Cδ are drawn in orange, and Oε1 is in red, and Nε2 is in blue. Anticodon nucleotides in skyblue are CUG.

Figure 25. This picture is the same as Figure 9 except for Asn. Cα, Cβ and Cγ are drawn in orange, and Oδ1 is in red and Nδ2 is in blue. Anticodon nucleotides in skyblue are GUU.

Figure 26. This picture is the same as Figure 9 except for Lys. Cα, Cβ, Cγ, Cδ and Cε are drawn in orange, and Nζ is in blue. Anticodon nucleotides in skyblue are CUU.

Figure 27. This picture is the same as Figure 9 except for His. Cα, Cβ, Cγ, Cδ2 and Cε1 are drawn in orange, and Nδ1 and Nε2 are in blue. Anticodon nucleotides in skyblue are GUG.

Figure 28. This picture is the same as Figure 9 except for Phe. Cα, Cβ, Cγ, Cδ1, Cδ2, Cε1, Cε2 and Cζ are drawn in orange. Anticodon nucleotides in skyblue are GAA

Figure 29. This picture is the same as Figure 9 except for Tyr. Cα, Cβ, Cγ, Cδ1, Cδ2, Cε1, Cε2 and Cζ are drawn in orange, and Oη are in red. Anticodon nucleotides in skyblue are GUA

Figure 30. This picture is the same as Figure 9 except for Cys. Cα and Cβ are drawn in orange, and Sγ is in green. Anticodon nucleotides in skyblue are GCA.

Figure 31. This picture is the same as Figure 9 except for Trp. Cα, Cβ, Cγ, Cδ1, Cδ2, Cε2, Cε3, Cζ2, Cζ3 and Cη2 are drawn in orange, and Nε1 are in blue. Anticodon nucleotides in skyblue are CCA

Figure 32. Stereoview of two adjacent anticodon loops, A-site tRNA is drawn in magenta and P-site tRNA in green. Anticodon nucleotides at the A-site are drawn in skyblue, while those at the P-site in orange. Messenger RNA for A-site is in blue, while that for P-site is in gold. The rest of the nucleotides for A-site tRNA is in brown, while that for P-site tRNA is in yellow. The peptides at A-site and P-site are Gly and Pro, respectively, and drawn in dark blue.

Figure 33. Stereoview of 6.5-fold symmetrical left-handed single-stranded polyuridine. This structure is also capable of base-pairing with the adjacent layer. The cavity in the layer is a little wider than that of 6-fold symmetrical one.

Figure 34. This picture is the same as Figure 33, but seen from a different direction in parallel with the axis of that of the left-handed helix.

Figure 35. Stereoview of α–helical structure of peptides drawn in blue and composed of L-amno acids, sequence of which is ProGlyAspAlaAlaSerVal.

Figure 36. Secondary structure of E. coli rRNA following the layout style of Gutell (1993). 16S rRNA along with TѰC-stem-loop of yeast Phe-tRNAPhe. 5’, C, 3’M, and 3’m represent 5’ domain for the region of 1-556, central domain for the region of 557-918, 3’-major domain for the region of 919-1396, and 3’-minor domain for the region of 1397-1542, respectively. Small numerals denote the residue numbers of the nucleotides indicated by the respective lines. The helices are numbered by large numbers as Brimacombe (1995).

Figure 37. Secondary structure of E. coli rRNA following the layout style of Gutell (1993). 5’ half of 23S rRNA. I, II and III represent domain numbers for the regions of 1-561, 562-1269 and 1270-1646, respectively. Numerals are as in Figure 36.

Figure 38. Secondary structure of E. coli rRNA following the layout style of Gutell (1993). 3’ half of 23S rRNA. IV, V and VI represent domain numbers for the regions of 1647-2014, 2015-2627 and 2628-2904, respectively.

 The α–sarcin-ricin stem-loop region (SRL), A-site finger, P-loop, and A-loop are in the nucleotide ranges from 2653 to 2667, from 879 to 898, from 2251 to 2253, and from 2552 to 2556, respectively. Numerals are as in Figure 36.

Figure 39. Secondary structure of 9S small subunit mitochondrial rRNA of Trypanosoma brucei. Residue number 1 is 1189th U EMBL database accession number X02547. Residues 493-506 UAUUAUUUAUAUUA in light blue are supposed to base-pair with the red residues in the SRL region of large subunit rRNA in Figure 40. Residues 515-519 UGUGU in green are supposed to base-pair with the T-loop of tRNAs. Large numbers 1, 2, 3, … represent helix numbers corresponding to those for E. coli secondary structure (Brimacombe, 1995), but some are missing (e.g. 6). 5’, C, and 3’m denote 5’-domain (the region of 1-556 in E. coli 16S rRNA), central domain (557-918 in E. coli 16S rRNA), and  3’-minor domain (1397-1542 in E. coli 16S rRNA), respectively, but 3’-major domain (919-1396 in E. coli 16S rRNA) is missing.

Figure 40. Secondary structure of 12S large subunit mitochondrial rRNA of Trypanosoma brucei. Residue numer 1 is 11th U of X02547. Residues 1130-1143 AAGUACGCAAGGAU in red are supposed to base-pair with the light blue residues of small subunit rRNA in Figure 39. Large numerals as in Figure 39. I, II, III, IV, V, and VI represent domain numbers for the regions corresponding to those of 1-561, 562-1269, 1270-1646, 1647-2014, 2015-2627, and 2628-2904 in E. coli 23S rRNA, respectively. The α-sarcin/ricin stemloop region (SRL), A site finger, P-loop, and A-loop correspond to those in E. coli 23S sequence in the nucleotide regions from 2653 to 2667, from 879 to 898, from 2251 to 2253, and from 2552 to 2556, respectively. Central loop is the highly-conserved nucleotide region surrounding tRNAs on the ribosome.

Figure1. Nucleotide sequence comparisons of two highly conserved regions from large subunit- and small subunit-rRNAs as well as the numbers of four typical sequence patterns of tRNA of respective species. (a) Eubacteria.

Figure2. Nucleotide sequence comparisons of two highly conserved regions from large subunit- and small subunit-rRNAs as well as the numbers of four typical sequence patterns of tRNA of respective species. (b) Archaea.

Figure3. Nucleotide sequence comparisons of two highly conserved regions from large subunit- and small subunit-rRNAs as well as the numbers of four typical sequence patterns of tRNA of respective species. (c) Chloroplast.

Figure4. Nucleotide sequence comparisons of two highly conserved regions from large subunit- and small subunit-rRNAs as well as the numbers of four typical sequence patterns of tRNA of respective species. (d) Eukarya.

Figure5. Nucleotide sequence comparisons of two highly conserved regions from large subunit- and small subunit-rRNAs as well as the numbers of four typical sequence patterns of tRNA of respective species. (e) Mitochondria.

Figure6. Universal rule of complementarities between 23S and 16S rRNAs and between 16S rRNA and tRNA.

Figure 47. Stereoview of main-chain representation for double-stranded helix formed between SRL-region, U2653-C2666, of 23S rRNA, which is drawn in magenta, and the region G1064-G1077of 16S rRNA in skyblue. The other region in yellow is G2645-C2652 and C2667-A2675 of H95. Some base pairs in H95, such as 2650-2652/2668-2670 are unfolded. Only main chains and base-pairing bases are drawn for simplicity. Red letters denote the locations of nucleotides, G1064, G1077, G2645, A2675, U2653, and C2667, respectively.

Figure 48. Virtual picture of stereoview for the near-cognate G-U wobble base pair, which is drawn by blue guanine and yellow uridine, at the first position of the A-site codon-anticodon helix. Mg2+ ion in green is kept between O2P atom of YG37 and O2 atom of 2’-O-methylated OMC32. In order to keep the wobble base pair, the base of U33 in magenta must be extended outward and destabilized because of the loss of hydrogen-bonding energy.

Figure 49. Stereoview of mRNA kinks used in Nagano (2015). Three codons at the A, P, and E sites, that are composed of all uridines, and two clusters of Mg2+ hexahydrate binding at the P/A- and E/P-kinks, by representing green big ball for Mg2+ ions and yellow big balls for water molecules. The sharp kinks are formed by binding one Mg2+ hexahydrate at one kink instead of two, which are observed in X-ray structures.

Figure 50. Stereoview of cognate A-site codon-anticodon helix and the ASL of Nagano (2015), in which U33 of the ASL and A1396 at the decoding site of 16S rRNA are drawn in magenta and brown, respectively. Significance of A1396 will be described in Figure 51. This picture is shown to look similar to that in Figure 48 as far as possible.

Figure 51. Stereoview of near-cognate A-site codon-anticodon helix and the ASL of Nagano (2015), in which G-U wobble base pair at the first base pairs of the A-site codon-anticodon helix is drawn in the same colors as those in Figure 48 and both U33 and A1396 are as those in Figure 50.

Figure 52. Stereoview of full-atom representation for the residues at the decoding site of 16S rRNA. U33 of P-site tRNA (green) is base-paired with A1398 (red), that of E-site tRNA (purple) base-paired with A1502 (pink), but U33 (magenta) at the recognition mode of A site (T site) is not base-paired. A1396 (brown) is far from the U33 base (magenta). G1401-C1501 (skyblue) is always formed. C1399-G1504 (gold) is formed in this case. This structure leads to rejection of either noncognate or near-cognate aa-tRNA from the A site. C1400 is drawn in yellow, while U1506, which starts h45, is in blue.

Figure 53. Stereoview of full-atom representation for the residues at the decoding site of 16S rRNA. This picture is very similar to Figure 52, but different from Figure 52 in the following two points. U33 at the A site (magenta) is base-paired with A1396 (brown), and U33 at the E site (purple) is not base-paired with A1502 (pink).

Figure 54. Stereoview of full-atom representation for the residues at the decoding site of 16S rRNA. This picture is also very similar to Figures 52 and 53, except that all three U33 at the A, P, and E sites are base-paired with A1396 (brown), A1398 (red), and A1502 (pink), respectively, but C1399-G1504 (gold) is broken.

Figure 55. Stereoview of main-chain representations of tRNAs and mRNA. P-site tRNA (green) in the center. Nucleotides are G2645-C2652 (white) and G2668-A2675 (white), with a label A indicating the location of G2645. The region of U2653-C2667 (yellow) is the SRL of 23S rRNA. Label B denotes the location of A76 of tRNA at the P site, mRNA (red) with Label C for its first residue. The following regions of 16S rRNA are shown in different colors: G1064-G1077 (skyblue), C1103-C1107 (cyan), G527-A533 (yellow tint), U1189-A1191 (pink), G1338-A1339 with bases (gold), A1408-A1420 (orange), G1480-A1493 (orange), U1506-G1529 (blue), C1399-G1504 pair with bases (violet), and A1396 (brown). Labels from D to P denote the locations of C545, G922, U956, U991, A1041, G1117, A1180, A1236, G1334, A1350, C1384, C1452, and U1542, respectively.

Figure 56. Stereoview of main-chain representations of transition-state three tRNAs, A-site tRNA (magenta), P-site tRNA (green), and E-site tRNA (purple). This structure corresponds to the local picture of the decoding site represented in Figure 54. Red label Q denotes the location of C805 of 16S rRNA

Figure 57. Stereoview of main-chain representations of three tRNA binding model, in which E-site tRNA (purple) takes an isolated L-form with its U33-folded anticodon loop. This structure corresponds to the local picture of the decoding site represented in Figure 53.

Figure 58. Stereoview of main-chain representations of three tRNA binding model, in which the three tRNAs are arranged as corresponding to the local picture of the decoding site in Figure 52, and aa-tRNA (magenta) takes an isolated L-form with its U33-folded anticodon loop.

Figure 59. Stereoview of main-chain representation for the conformations of the 3’-end regions of tRNAs surrounded by the nucleotides at the H80 (P-loop) and H88. It is known that C2422 can bind to the 3’-end adenine base (A76) of deacylated tRNA at either P or E site. The loop of H88, A2407-G2410, is tightly bound with C413-U416 at the loop of H22, which is drawn in yellow, and works as widely movable long rod, by fixing its one end at the position of G424 on the 50S subunit, between the 3’-end region of P-site tRNA (green) in the PRE state, and that of E-site tRNA (purple) in the POST state. This structure of 3’-end regions of two tRNAs on the 50S subunit in the PRE state. The regions of G1-U8 and A66-A76 of A-site tRNA (magenta), G1-U8 and A66-A76 of P-site tRNA (green), G2246-C2258 (H80) (red), C2395-G2421 (H88) (cyan), G406-C421 (H22) (yellow) of 23S rRNA and the other associated regions (greyish white) are drawn. This corresponds to the intermediate structure in the A/P and P/E hybrid states.

Figure 60. Stereoview of main-chain representation for the conformations of the 3’-end regions of tRNAs surrounded by the nucleotides at the H80 (P-loop) and H88. This structure of 3’-end regions of two tRNAs on the 50S subunit in the POST state. The regions of G1-U8 and A66-A76 of P-site tRNA (green), G1-U8 and A66-A76 of E-site tRNA (purple), G2246-C2258 (H80) (red), C2395-G2421 (H88) (cyan), G406-C421 (H22) (yellow) of 23S rRNA and the other regions (greyish white) are drawn. This corresponds to the structure in the classical P/P and E/E states.

Figure 61. Stereoview of elongation factors during the course of A-site accommodation and translocation with GTP hydrolysis. Main-chan representation of GTP molecule (red) at the border of RSL from U2656 to G2661 (yellow) and 5’-half of h35 of 16S rRNA from G1069 to G1072 (skyblue)

Figure 62. Stereoview of elongation factors during the course of A-site accommodation and translocation with GTP hydrolysis. The same model in Figure 61 is shown from the other direction that is from the 3’-end of P-site tRNA (green) toward the mRNA. When the codon-anticodon interaction is that of cognate, the conformations of the ASL and the elbow region of T-site tRNA (magenta) induces the GTP hydrolysis, ending at the conformation of A-site tRNA as shown in Figures 56 and 57. Blue ribbon, and the other nucleotide chains represent the peptide chain of EF-Tu from A1 to E405 with VDHGKTT (20-26) missing, RSL region (C2652-C2667 in yellow), 5’-side of 16S rRNA (G1064-G1077 in skyblue), T-site tRNA (A-U8, A14-U15, 5MC49, U68, U72 and A76 in magenta), and P-site tRNA (G45-C56 and C61-G65 in green), respectively. The regions of T- and P-site tRNAs are only shown for those in close contact with EF-Tu. The region of U72-A76 in Trp-tRNATrp (brown) is taken to connect the main body of T-site tRNA and the EF-Tu polypeptide regions (ribbon) stretching toward GTP binding site by A1-R190 and P-site tRNA by G191-E405.

Figure 63. Stereoview of elongation factors during the course of A-site accommodation and translocation with GTP hydrolysis. The location and conformation of EF-Tu is shown after the accommodation of A-site tRNA is finished, EF-Tu leaves the ribosome immediately. The rest of the model is the same as that of Figure 57.

Figure 64. Stereoview of elongation factors during the course of A-site accommodation and translocation with GTP hydrolysis. The ancestral form of EF-G bound to the PRE state ribosome is shown. The body of tRNA in gold corresponds to the domain IV of EF-G. The rest of the ancestral EF-G is the same as that of EF-Tu, which appeared in Figure 62. Since A-site is occupied by a big body of tRNA, the location of GTP binding to the border of RSL of 23S rRNA and 5’-half of h35 is shifted by as far as 27 Å from the P-site tRNA.

Figure 65. Comparison of amino acid sequences of large subunit ribosomal proteins L2, L3 and L4. L2 sequences are taken for 1) E. coli, 2) Paenibacillus pabuli (P. pab) and 3) Thermus thermophillus thermophillus (T. ther), L3 sequences are for 1) E. coli, 2) Alicyclobacillus sendaiensis (A. sen), and 3) T. ther and L4 sequences are for 1) E. coli, 2) Bacillus vallismortis (B. vall), and 3) T. ther . Common amino acids at the corresponding position with that of E. coli sequence are represented by blank spaces. Amino acid residues, which are common in three species and basic such as R or K, are drawn in blue, those, which are common in three species and acidic such as D or E, are drawn in red, and those, which are common in three sequences and hydrophobis such as V, or L, or I, or M, or F, are drawn in brown.

Figure 66. Binding site of ribosomal protein S2 on the present model. Blue ribbon represents the location of the binding protein.

Figure 67. Binding site of ribosomal protein S3 on the present model. Blue ribbon represents the protein.

Figure 68. Binding site of ribosomal protein S4 on the present model. Blue ribbon represents the protein.

Figure 69. Binding site of ribosomal protein S5 on the present model. Blue ribbon represents the protein.

Figure 70. Binding site of ribosomal protein S6 on the present model. Blue ribbon represents the protein.

Figure 71. Binding site of ribosomal protein S7 on the present model. Blue ribbon represents the protein.

Figure 72. Binding site of ribosomal protein S8 on the present model. Blue ribbon represents the protein.

Figure 73. Binding site of ribosomal protein S9 on the present model. Blue ribbon represents the protein.

Figure 74. Binding site of ribosomal protein S10 on the present model. Blue ribbon represents the protein.

Figure 75. Binding site of ribosomal protein S11 on the present model. Blue ribbon represents the protein.

Figure 76. Binding site of ribosomal protein S12 on the present model. Blue ribbon represents the protein.

Figure 77. Binding site of ribosomal protein S13 on the present model. Blue ribbon represents the protein.

Figure 78. Binding site of ribosomal protein S14 on the present model. Blue ribbon represents the protein.

Figure 79. Binding site of ribosomal protein S15 on the present model. Blue ribbon represents the protein.

Figure 80. Binding site of ribosomal protein S16 on the present model. Blue ribbon represents the protein.

Figure 81. Binding site of ribosomal protein S17 on the present model. Blue ribbon represents the protein.

Figure 82. Binding site of ribosomal protein S18 on the present model. Blue ribbon represents the protein.

Figure 83. Binding site of ribosomal protein S19 on the present model. Blue ribbon represents the protein.

Figure 84. Binding site of ribosomal protein S20 on the present model. Blue ribbon represents the protein.

Figure 85. Binding site of ribosomal protein THX on the present model. Blue ribbon represents the protein.

Figure 86. Binding site of ribosomal protein L1 on the present model. Blue ribbon represents the protein.

Figure 87. Binding site of ribosomal protein L2 on the present model. Blue ribbon represents the protein.

Figure 88. Binding site of ribosomal protein L3 on the present model. Blue ribbon represents the protein.

Figure 89. Binding site of ribosomal protein L4 on the present model. Blue ribbon represents the protein.

Figure 90. Binding site of ribosomal protein L5 on the present model. Blue ribbon represents the protein.

Figure 91. Binding site of ribosomal protein L6 on the present model. Blue ribbon represents the protein.

Figure 92. Binding site of ribosomal protein L9 on the present model. Blue ribbon represents the protein.

Figure 93. Binding site of ribosomal protein L11 on the present model. Blue ribbon represents the protein.

Figure 94. Binding site of ribosomal protein L13 on the present model. Blue ribbon represents the protein.

Figure 95. Binding site of ribosomal protein L14 on the present model. Blue ribbon represents the protein.

Figure 96. Binding site of ribosomal protein L15 on the present model. Blue ribbon represents the protein.

Figure 97. Binding site of ribosomal protein L16 on the present model. Blue ribbon represents the protein.

Figure 98. Binding site of ribosomal protein L18 on the present model. Blue ribbon represents the protein.

Figure 99. Binding site of ribosomal protein L19 on the present model. Blue ribbon represents the protein.

Figure 100. Binding site of ribosomal protein L20 on the present model. Blue ribbon represents the protein.

Figure 101. Binding site of ribosomal protein L21 on the present model. Blue ribbon represents the protein.

Figure 102 Binding site of ribosomal protein L22 on the present model. Blue ribbon represents the protein.

Figure 103. Binding site of ribosomal protein L23 on the present model. Blue ribbon represents the protein.

Figure 104. Binding site of ribosomal protein L24 on the present model. Blue ribbon represents the protein.

Figure 105. Binding site of ribosomal protein L25 on the present model. Blue ribbon represents the protein.

Figure 106. Binding site of ribosomal protein L27 on the present model. Blue ribbon represents the protein.

Figure 107. Binding site of ribosomal protein L29 on the present model. Blue ribbon represents the protein.

Figure 108. Binding site of ribosomal protein L30 on the present model. Blue ribbon represents the protein.

Figure 109. Binding site of ribosomal protein L31 on the present model. Blue ribbon represents the protein.

Figure 110. Binding site of ribosomal protein L35 on the present model. Blue ribbon represents the protein.

Figure 111. Binding site of ribosomal protein L36 on the present model. Blue ribbon represents the protein.

Figure 112. This picture shows a comparison of amino acid sequences of effector region for 4 GTPases, EF-Tu (and eEF-1), EF-G (and eEF-2), termination factor 3 (RF3), and initiation factor 2 (IF-2) (and aIF-2). The amino acid sequences near GTP binding site and highly conserved and acidic such as D and E are drawn in red, those of basic such as K or R are in blue, and those of the rest are in green. The abbreviated species name, the species name, the accession file name, and thir residue number range of the amino acid sequence are obtained from Swiss Protein Sequence Data Bank (PIR): (1) eubacterial elongation factor EF-Tu; E. coli, Escherichia coli EFECT 17-65, Taqua, Thermus aquaticus S00229 17-166. Hinfl, Haemophilus influenza E64078 17-65, Pfalc, Plasmodium falciparum plastid S72277 17-65, elEgrat, Euglena gracilis chloroplast EFEGT 17-66, clPpurp, Porphyra purpurea chloroplast S73208 17-65, mtCeleg, Caemorhabditis elegans mitochondria T37273 50-98, mtScere, Saccharomyces cerevisiae mitochondria EFBYT 53-101. (2) eukaryotic elongation factor eEF-1α; Scere, S. cerevisiae EFBY1A 12-75. Tbruc, Trypanosoma brucei A54760 12-75, Pfalc, P. falciparum S21909 12-75, Zmays, Zea mays S66339 12-75. Tpyri, Tetrahymena pyriformis A49171 13-76, Hsapi, Homo sapiens EFHU1 12-75. (3) eubacterial elongation factor EF-G; E. coli, E. coli EFECG 15-65, S6803, Synechocytis sp. PCC 6803 S75863 15-65, T. aqua, T. aquaticus EFTWG 17-67, Dradi, Deinococcus radiodurans E75536 18-68, clGmax, Glycine max chloroplast S35701 105-155, mtScere, S. cerevisiae mitochondria S43748 46-96, mtRnorv, Rattus norvegius mitochondria S40780 52-105. (4) eukaryotic elongation factor eEF-2; Scere, S. cerevisiae A41778 24-72, Bvulg, Beta vulgaris T14579 24-72, Athal, Arabidopsis thaliana A96602 27-75, Celeg, C. elegans A40411 24-72. Dmela, Drosophila melanogaster S05988 24-72. Hsapi, H. sapiens EFHU2 24-72. (5) eubacterial peptide chain release factor RF-3; Ecoli, E. coli E91295 18-72. Sente, Salmonella enterica AD1072 18-72, Paeru, Pseudomonas aeruginosa B83159 16-70, B.APS, Buchnera sp. APS C84993 18-72, Linno, Listeria innocua AB1556 17-71, Llact, Lactococcus lactis E86668 15-69, Spneu, Streptococcus pneumoniae D97921 15-69, N7120, Nostoc sp. PCC 7120 AC2353 21-75, Saure, Staphylococcus aureus D89870 15-69, Atume, Agrobacterium tumefaciens AC2615 17-71, Bmeli, Brucella melitensis AB3539 15-69, Ceres, Caulobacter crescentus B87382 17-71. (6) eubacterial initiation factor IF-2; Ecoli, E. coli FIEC2 396-463, Bsubt, Bacillus subtilis A35269 224-291, Saure, S. aureus H89900 214-281, clPpurp, P. purpurea chloroplast S73178 268-335, mtScere, S. cerevisiae mitochondria S66706 150-219, mtSpombe, Schizosaccharomyces pombe mitochondria T39351 176-245. (7) archeal initiation factor aIF-2; Mther, Methanobacterium thermoautotropicum E69132 10-95, Aflug, Archaeoglobus fulgidus H69345 18-100, Hsali, Halobacterium salinarum T43849 20-103.