Reviewer 1: Dr. Henri Grosjean (nominated by Dr. P. Lopez-Garcia)
In this manuscript, the authors hypothesize that the specific functions assigned to the 2′-3′ hydroxyls during peptide bond formation on the ribosome has co-evolved with the mechanism of tRNA aminoacylation and editing activities by the 2 classes of aminoacyl-tRNA synthetases (aaRSs), and with the isomerase activity of EF-Tu employing a mixture of the 2′(3′)-aminoacyl-tRNA isomers as substrates. Their main idea of the paper is that the true catalysts of peptide bond formation on the ribosome are the aminoacylated tRNA molecules, the aaRSs and the PTC of ribosome mainly allowing the proper positioning of the aa-tRNAs with the respective active sites. In other words, the tRNA molecule merely functions as a ribozyme, an idea that fits with the now generally admitted hypothesis that ancient protein synthesis machinery was governed by RNA. This paper is a logical prolongation of an earlier work (J. Theor Biol-2004) more focused on the coevolution hypothesis between amino acid biogenesis, aaRSs and the genetic code. These two papers complete each other very well. The present manuscript is clearly written, and while no experimental evidences are provided, the hypothesis is convincing.
Minor remarks: Page 8 GCA ancient codon? This should be developing a bit because the argument is not obvious. Page 9 > > 20° should be 20°C of course. Page 15, cys-editing should be written cis-editing (this mistake occurs 3 times in the text).
Authors’ response: We accepted all the remarks of Reviewer 1, and corrected the text accordingly. As to the antiquity of the GCA codons, we added a few sentences at the end of that discussion, to clarify our arguments.
Reviewer 2: Prof. Manuel Santos (nominated by Prof. Yitzhak Pilpel)
In this hypothesis paper Mark Safro and Liron Klipcan put forward the hypothesis that the functions of the 2′and 3′ hydroxyls of the conserved tRNA 3′-terminal adenosine (A76) observed during peptide bond formation at the peptidyl transferase centre (PTC) of the ribosome co-evolved with the two modes of attack on the aminoacyl-adenylate carbonyl typical of the two classes of aaRSs and also with the isomerase activity of EF-Tu. The authors recognize the fundamental roles of the 3′-OH and 2′-OH groups in protein synthesis and develop their arguments with several examples that highlight the relevance of the 2′-OH and 3′-OH chemistry at the amino acid activation, editing, transfer to the elongation factor and PTC levels. This is an expected outcome of the 2′-OH and 3′-OH multilayered chemistry, it would be surprising if it worked in a different manner. The value of this highly descriptive hypothesis paper that reviews old concepts is that it highlights the overlooked chemistry of the 2′-OH and 3′-OH, bringing it to current debate. This is important and sufficient to recommend its publication. However, there are several aspects of the paper that should be improved before publication, namely:
1. The first part of the manuscript (including the abstract) is poorly written and should be improved. There are many grammatical errors along the manuscript.
2. On page-4, third paragraph, the authors state that “the ribosome is a spectacular fossil artefact”. Looking at the ribosome as a fossil artifact is simply wrong. This should be changed to make to sentence clear.
3. The 3 subheadings dedicated to the aminoacylation reaction, the aaRS classes and the early role of Class-II aaRS in genetic code translation should be fused into a single subheading and the text shortened. It is too descriptive and repetitive.
4. Page-14, second paragraph. The sentence “….transtion from statistically encoded proteins to error-prone translation” should be clarified. It does not make sense.
5. Page -14, third paragraph, additional references should be included. This is a highly speculative paragraph that could be improved.
Quality of written English: Needs some language corrections before being published.
Authors’ response: As to the criticism of the sentences on pages 4 and 14 we formulated them in more clear form by modifying the text thus: a) the sentence on page 4 now reads, “The ribosome is an ancient molecular machine governed by RNA”; b) the corrected sentence on page 14 now reads, “A cornerstone in the evolution of the genetic code entails the transition from early proteins that may have been inherently statistical; that is, one of several stereochemically analogous amino acids might have been brought to a given position in the polypeptide chain, to protein with unique sequences and 3D-structures.” According to the referee’s request we added references within the third paragraph on page 14 and 15.
The authors agreed with the referee’s suggestion to improve the first part of the manuscript by combining three subheadings of the manuscript dedicated to the aminoacylation reaction, the aaRS classes, and the early role of Class-II aaRS in genetic code translation, into a single section. We fully rewrote this part of the manuscript. While we agree with the reviewer that the introduction should contain a more comprehensive description of the translational apparatus, we would note that the modern system of genetic code translation, even at its simplest, is an extremely complex process, and includes a fair number of macromolecular components. A detailed description of even its major elements would increase the length of the manuscript dramatically.
Reviewer 3: Dr. Koonin E.V.
This article discusses a problem that is both enormously important and staggeringly hard, namely the origin of the modern-type translation system. The authors suggest propose several interesting ideas and make remarkable inferences. To me, the most striking point is that tRNAs effectively act as ribozymes in the modern translation system, both in the aminoacylation and in the peptidyltransferase reactions. Even if not entirely new, this is a startling conclusion, and I find it regrettable that the authors explicitly mention it only in passing, in the Concluding Remarks. The main point of the article, however, is the hypothesis that modern-type translation started with aminoacylation of 3′-OH of A76 in tRNAs that is facilitated by Class II aaRS. Subsequently, under this scenario, Class I aaRS that initially aminoacylate at 2′-OH of A76, followed by isomerization facilitated by EF-Tu, were added to the system. The idea that 3′-OH aminoacylation is primordial certainly is plausible, given that this is the final form in which aminoacyl-tRNAs participate in peptide synthesis. Furthermore, the association of Class II aaRS with chemically simple and supposedly primordial amino acids is compatible with the primacy of this class. However, the evidence of this primacy from protein sequence and structure comparison is weak at the very best. There is just as good a reason to believe that Class II aaRS evolved from coenzyme biosynthesis enzymes (biotin synthetase, same superfamily in SCOP) as there is for class I aaRS, it is just less well publicized. Furthermore, the fact that the substrate-binding domain of class I aaRS belongs to one of the most common, simple protein folds (the Rossmann fold) whereas Class II enzymes adopt a more rare and complex fold, might be used as argument for the primacy of Class I. Moreover, the author repeatedly imply that the transition from statistical peptide synthesis to modern type translation was brought about by the recruitment and diversification of the aaRS. This is extremely unlikely to be the case because both classes of the aaRSs seem to be rather late arrivals in the evolution of the respective protein folds, their emergence being antedated by extensive protein evolution [1,2]. The same holds for EF-Tu which belongs to a small branch within the huge GTPase superfamily . Thus, counterintuitive as that might seem, apparently, there was a high-fidelity RNA-based translation system, and one would think that the roots of the key features of modern translation should be sought there. In that regard, it is very strange that the authors do not examine the properties of the small aminoacylating ribozymes which are among the most remarkable molecules that are relevant for the origin of translation. The GUGGC-3′ ribozyme indeed aminoacylates at 3′-OH  which is fully compatible with the primacy of this reaction.
Thus, the hypothesis proposed by the authors in itself is indeed plausible and deserves publication and discussion. However, in the present manuscript, much of the argument is missing, flawed, muddled or tangentially relevant. In addition to the major issues outlined above, I find the discussion of cysteine incorporation in Archaea (which the authors consider to be ancestral without any good reason) as well as the mechanism of PheRS to be largely irrelevant and more confusing than enlightening.
1. Aravind L, Mazumder R, Vasudevan S, Koonin EV: Trends in protein evolution inferred from sequence and structure analysis. Curr Opin Struct Biol. 2002, 12:392-399.
2. Aravind L, Anantharaman V, Koonin EV: Monophyly of class I aminoacyl tRNA synthetase, USPA, ETFP, photolyase, and PP-ATPase nucleotide-binding domains: implications for protein evolution in the RNA. Proteins 2002, 48:1-14.
3. Leipe DD, Wolf YI, Koonin EV, Aravind L: Classification and evolution of P-loop GTPases and related ATPases. J Mol Biol 2002, 317(1):41-72.
4. Yarus M: The meaning of a minuscule ribozyme. Philos Trans R Soc Lond B Biol Sci. 2011, 366:2902-2909.
a. Reviewer’s comment: “..the association of Class II aaRS with chemically simple and supposedly primordial amino acids is compatible with the primacy of this class. However, the evidence of this primacy from protein sequence and structure comparison is weak at the very best. There is just as good a reason to believe that Class II aaRS evolved from coenzyme biosynthesis enzymes (biotin synthetase, same superfamily in SCOP) as there is for Class I aaRS, it is just less well publicized. Furthermore, the fact that the substrate-binding domain of Class I aaRS belongs to one of the most common, simple protein folds (the Rossmann fold) whereas Class II enzymes adopt a more rare and complex fold, might be used as argument for the primacy of Class I”.
Answer: Indeed, we agree that aaRSs associated with class I and II aaRSs evolved at a time when some proteins and canonical folds were already well-established. These ancient proteins might be associated with coenzyme biosynthesis enzymes such as biotin synthetase, or with biosynthesis of fatty acid derivatives, for example. We previously showed that all structural domains of biotin synthetase have structural homologs in multi-domain β-subunit of PheRS. Remarkable similarity when all structural domains of one multi-domain protein appear to be constituents of the other multidomain protein supports a concept of a common ancestor for two different enzymes [1,2].
In our discussion, we entertain the proposal that class II aaRSs were the first to replace ribozyme-based aminoacylation. Considerable evidence exists in favor of this point: 1) the amino acids formed in the Miller experiment are mostly class II-related; 2) the reconstructed chronology of amino acids introduction into the genetic code, as presented by E. Trifonov , strongly suggests the association of early amino acids with class II; 3) the so-called, more ancient “second genetic code”, located in the stem-loop of the tRNA, is primarily recognized by class II aaRSs; 4) our findings  suggest that organization of amino acid biosynthetic pathways, and clustering of aaRSs into different classes are intimately related to one another. A plausible explanation for such a relationship is dictated by early link between aaRSs and amino acid biosynthetic proteins. The aaRSs’ catalytic cores are highly relevant to the ancient metabolic reactions, namely to amino acid and cofactors biosynthesis. In particular it has been shown that class II aaRSs mostly associated with the primordial amino acids, while class I aaRSs are usually related to amino acids that evolved at a later stage . The statement regarding the simplicity and structural priority of the Rossmann fold, at least, is non-trivial. For example it was hypothesized that aaRSs of two classes were originally associated with one common tRNA molecule [5,6]. Additionally, Carter and Duax in 2002 reported that complementary fragments of the specific DNA region in Achlya klebsiana code for proteins (not aaRSs) that have the same folds as class I and II synthetases .
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b. Reviewer’s comment: “…I find the discussion of cysteine incorporation in Archaea (which the authors consider to be ancestral without any good reason) as well as the mechanism of PheRS to be largely irrelevant and more confusing than enlightening”.
Answer: Genome-wide analysis revealed that cysteine content was dramatically increased (a so- called “gainer” amino acid) only after the existence of LUCA, suggesting late incorporation of cysteine into the genetic code [8,9]. Indeed, in organisms using the standard CysRS-tRNACys pathway of aminoacylation, this probably is the case. However, our concept is that high cysteine content in methanogenic Archaea (considered being very ancient) and the way it forms Cys-tRNACys (via a tRNA-dependent pathway of cysteine biosynthesis, using L-phosphoserine as a precursor, and the class II aaRS SepRS ), suggests that cysteine was abundant in some ancient organisms. We used this example as an indication of the antiquity of tRNA-dependent pathways in such organisms. This point of view was supported very recently by results published by Zhang et. al. : “These results indicate that tRNA-dependent Cys biosynthesis appeared 500 million years earlier (~3.5 Ga) than the tRNA-independent counterparts (~3.0 Ga), supporting a previous opinion that tRNA-dependent Cys biosynthesis had a very ancient origin (Klipcan et al., 2008)”.
8) Jordan IK, Kondrashov FA, Adzhubei IA, Wolf YI, Koonin EV, Kondrashov AS, Sunyaev S. A universal trend of amino acid gain and loss in protein evolution. Nature. 2005, 433:633-8.
9) Brooks DJ, Fresco JR. Increased frequency of cysteine, tyrosine, and phenylalanine residues since the last universal ancestor. Mol Cell Proteomics. 2002, 1:125-31.
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11) Zhang H-Y, Qin T, Jiang Y-Y, Caetano-Anollés G. Structural phylogenomics uncovers the early and concurrent origins of cysteine biosynthesis and iron-sulfur proteins. J. Biom. Str. Dynamics, 2012; 30:542–545.
c) Reviewer’s comment: “…This is extremely unlikely to be the case because both classes of the aaRS seem to be rather late arrivals in the evolution of the respective protein folds, their emergence being antedated by extensive protein evolution [12,13]. The same holds for EF-Tu which belongs to a small branch within the huge GTPase superfamily .
Answer: We completely agree with the reviewer’s remark that the RNA based translation system was already well-established; thus, could be active without EF-Tu [15,16] and based on aaRSs ribozymes.
12) Aravind L, Mazumder R, Vasudevan S, Koonin EV: Trends in protein evolution inferred from sequence and structure analysis. Curr Opin Struct Biol. 2002, 12:392-399.
13) Aravind L, Anantharaman V, Koonin EV: Monophyly of class I aminoacyl tRNA synthetase, USPA, ETFP, photolyase, and PP-ATPase nucleotide-binding domains: implications for protein evolution in the RNA. Proteins. 2002, 48:1-14.
14) Leipe DD, Wolf YI, Koonin EV, Aravind L: Classification and evolution of P-loop GTPases and related ATPases. J Mol Biol. 2002, 317:41-72.
15) Gavrilova LP and Spirin AS: Stimulation of "non-enzymic" translocation in ribosomes by p-chloromercuribenzoate. FEBS Lett. 1971, 17:324-326.
16) Gavrilova LP, Kostiashkina OE, Koteliansky VE, Rutkevich NM and Spirin AS: Factor-free (“Non-enzymic”) and factor-dependent systems of translation of polyuridylic acid by Escherichia coli ribosomes. J Mol Biol. 1976. 101:537-552.
This referee made remarks regarding the grammatical imprecision, misspellings, and quality of the written English. We accepted all his suggestions, and made corrections accordingly. The manuscript was extensively edited by a scientific editor.