Revisiting the operational RNA code for amino acids: Ensemble attributes and their implications

It has been suggested that tRNA acceptor stems specify an operational RNA code for amino acids. In the last 20 years several attributes of the putative code have been elucidated for a small number of model organisms. To gain insight about the ensemble attributes of the code, we analyzed 4925 tRNA sequences from 102 bacterial and 21 archaeal species. Here, we used a classification and regression tree (CART) methodology, and we found that the degrees of degeneracy or specificity of the RNA codes in both Archaea and Bacteria differ from those of the genetic code. We found instances of taxon-specific alternative codes, i.e., identical acceptor stem determinants encrypting different amino acids in different species, as well as instances of ambiguity, i.e., identical acceptor stem determinants encrypting two or more amino acids in the same species. When partitioning the data by class of synthetase, the degree of code ambiguity was significantly reduced. In cryptographic terms, a plausible interpretation of this result is that the class distinction in synthetases is an essential part of the decryption rules for resolving the subset of RNA code ambiguities enciphered by identical acceptor stem determinants of tRNAs acylated by enzymes belonging to the two classes. In evolutionary terms, our findings lend support to the notion that in the pre-DNA world, interactions between tRNA acceptor stems and synthetases formed the basis for the distinction between the two classes; hence, ambiguities in the ancient RNA code were pivotal for the fixation of these enzymes in the genomes of ancestral prokaryotes.     

Visualization by cryo-electron microscopy of genomic RNA that binds to the protein capsid inside bacteriophage MS2

The icosahedrally symmetrized structure of bacteriophage MS2 as determined by cryo-electron microscopy (EM) reveals the presence of genomic RNA that attaches to coat-protein dimers. Earlier X-ray diffraction studies revealed similar interactions between the unique operator hairpin of the MS2 genomic RNA and the coat-protein dimer. This observation leads us to conclude that not only the operator, but also many other RNA sequences in the genome of MS2, are able to bind to the coat-protein dimer. A substantial number of potential coat-protein-dimer binding sites are present in the genome of MS2 that can account for the observed RNA densities in the EM map. Moreover, it appears that these stem-loop structures are able to bind in a similar fashion to the coat protein dimer as the wild-type operator hairpin. The EM map also shows additional density between the potential operator-binding sites, linking the RNA stem-loops together to form an icosahedral network around the 3 and 5-fold axes. This RNA network is bound to the inside of the MS2 capsid and probably influences both capsid stability and formation, supporting the idea that capsid formation and RNA packaging are intimately linked to each other.     

The MS2 coat protein shell is likely assembled under tension: a novel role for the MS2 bacteriophage A protein as revealed by small-angle neutron scattering

Recombinant forms of the bacteriophage MS2 and its RNA-free (empty) MS2 capsid were analyzed in solution to determine if RNA content and/or the A (or maturation) protein play a role in the global arrangement of the virus protein shell. Analysis of the (coat) protein shell of recombinant versions of MS2 that lack the A protein revealed dramatic differences compared to wild-type MS2 in solution. Specifically, A protein-deficient virus particles form a protein shell of between 31(+/-1) A and 37(+/-1) A. This is considerably thicker than the protein shell formed by either the wild-type MS2 or the RNA-free MS2 capsid, whose protein shells have a thickness of 21(+/-1) A and 25(+/-1) A, respectively. Since the A protein is known to separate from the intact MS2 protein shell after infection, the thin shell form of MS2 represents the pre-infection state, while the post-infection state is thick. Interestingly, these A protein-dependent differences in the virus protein shell are not seen using crystallography, as the crystallization process seems to artificially compact the wild-type MS2 virion. Furthermore, when the A protein is absent from the virus shell (post-infection), the process of crystallization exerts sufficient force to convert the protein shell from the post-infection (thick) state to the pre-infection (thin) conformation. In summary, the data are consistent with the idea that RNA content or amount does not affect the structure of the MS2 virus shell. Rather, the A protein influences the global arrangement of the virus coat dramatically, possibly by mediating the storage of energy or tension within the protein shell during virus assembly. This tension may later be used to eject the MS2 genomic RNA and A protein fragments into the host during infection.     

A new RNA vaccine platform based on MS2 virus-like particles produced in Saccharomyces cerevisiae

mRNA vaccines are potentially attractive alternatives to DNA vaccines more often discussed, as they are generally considered safer than their DNA counterparts. The major limitations on the potency of RNA vaccines are their instability and inability to spread in vivo. Virus-like particles (VLPs) based on the bacteriophage MS2 have demonstrated remarkably high stability and may provide an improved platform for RNA-based genetic vaccination. However, no in vivo study of an MS2 VLP-mediated RNA vaccine has been reported. Therefore, we developed a model vaccine wherein MS2 VLPs packaging HIV-1 gag mRNAs (1544 bases) were produced in Saccharomyces cerevisiae, and then, used to immunize BALB/c mice. Serological analyses showed that antigen-specific antibody responses were elicited by immunization. These findings suggest that MS2 VLPs can be used in the design and construction of novel and safe phage-based mRNA delivery vectors.     

MS2噬菌体衣壳蛋白与包装位点结合特异性及其生物学应用进展

MS2噬菌体为正义单链RNA噬菌体,基因组含有3569个核苷酸,编码成熟酶蛋白、衣壳蛋白、复制酶蛋白和裂解蛋白。MS2噬菌体复制酶编码基因5'端一个由19个碱基组成的茎环结构(又称包装位点)是衣壳蛋白二聚体与RNA相互作用的部位,二者相互作用形成的复合物是启动噬菌体自我包装的信号。MS2噬菌体衣壳蛋白与包装位点结合的特异性已被应用于RNA病毒核酸检测的标准物质、校准品和质控品的研究,实时动态监测活细胞内RNA的运动,以及RNA体内递送载体的研究等领域。     

Codon:Anticodon and anticodon:Anticodon interaction. Evaluation of equilibrium and kinetic parameters of complexes involving a G:U wobble

In order to learn about the effect of the G:U wobble interaction we characterized the codon:anticodon binding between triplets: UUC, UUU and yeast tRNA^Phe (anticodon GmAA) as well as the anticodon:anticodon binding between Escherichia coli tRNA^Glu₂, E. coli tRNA^Lys (anticodons: mam⁵s²UUC, and mam⁵s²UUU, respectively) and tRNA^Phe from yeast and E. coli (anticodon GAA) using equilibrium fluorescence titrations and temperature jump measurements with fluorescence and absorption detection. The difference in stability constants between complexes involving a G:U pair rather than a usual G:C basepair is in the range of one order of magnitude and is mainly due to the shorter lifetime of the complex involving G:U in the wobble position. This difference is more pronounced when the codon triplet is structured, i.e., is built in the anticodon loop of a tRNA. The reaction enthalpies of the anticodon:anticodon complexes involving G:U mismatching were found to be about 4 kcal/mol smaller, and the melting temperatures more than 20°C lower, than those of the corresponding complexes with the G:C basepair. The results are discussed in terms of different strategies that might be used in the cell in order to minimize the effect of different lifetimes of codon-tRNA complexes. Differences in these lifetimes may be used for the modulation of the translation efficiency.     

Block structure and stability of the genetic code

It is known that different codons may be unified into larger groups related to the hierarchical structure, approximate hidden symmetries, and evolutionary origin of the universal genetic code. Using a simplified evolutionary motivated two-letter version of genetic code, the general principles of the most stable coding are discussed. By the complete enumeration in such a reduced code it is strictly proved that the maximum stability with respect to point mutations and shifts in the reading frame needs the fixation of the middle letters within codons in groups with different physico-chemical properties, thus, explaining a key feature of the universal genetic code. The translational stability of the genetic code is studied by the mapping of code onto de Bruijn graph providing both the compact visual representation of mutual relationships between different codons as well as between codons and protein coding DNA sequence and a powerful tool for the investigation of stability of protein coding. Then, the results are extended to four-letter codes. As is shown, the universal genetic code obeys mainly the principles of optimal coding. These results demonstrate the hierarchical character of optimization of universal genetic code with strictly optimal coding being evolved at the earliest stages of molecular evolution. Finally, the universal genetic code is compared with the other natural variants of genetic codes.     

The Impact of Viral RNA on the Association Rates of Capsid Protein Assembly: Bacteriophage MS2 as a Case Study

A large number of single-stranded RNA viruses, which form a major class of all viruses, co-assemble their protein container and their genomic material. The multiple roles of the viral genome in this process are presently only partly understood. Recent experimental results indicate that RNA, in addition to its function as a repository for genetic information, could play important functional roles during the assembly of the viral protein containers. An investigation of the impact of genomic RNA on the association of the protein subunits may therefore provide further insights into the mechanism of virus assembly. We study here the impact of viral RNA on the association rates of the capsid proteins during virus assembly. As a case study, we consider the viral capsid of bacteriophage MS2, which is formed from 60 asymmetric (AB) and 30 symmetric (CC) protein dimers. Using Brownian dynamics simulations, we investigate the effect of the binding of an RNA stem-loop (the translational repressor) on the association rates of the capsid protein dimers. Our analysis shows that translational repressor binding results in self-association of AB dimers being inhibited, whilst association of AB with CC dimers is greatly enhanced. This provides an explanation for experimental results in which an alternating assembly pattern of AB and CC dimer addition to the growing assembly intermediate has been observed to be the dominant mode of assembly. The presence of the RNA hence dramatically decreases the number of dominant assembly pathways and thereby reduces the complexity of the self-assembly process of these viruses.     

A possible circular RNA at the origin of life

The increasing volume of sequenced genomes and the recent techniques for performing in vitro molecular evolution have rekindled the interest for questions on the origin of life. Nevertheless, a gap continues to exist between the research on prebiotic chemistry and molecule generation, on one hand, and the study of molecular fossils preserved in genomes, on the other. Here we attempt to fill this gap by using some assumptions about the prebiotic scenario (including a strong stereochemical basis for the genetic code) to determine the RNA sequences more likely to appear and subsist. A set of minimal RNA rings is exhaustively determined; a subset of them is then selected through stability arguments, and a particular ring ("AL ring") is finally singled out as the most likely winner of this prebiotic game. The rings happen to have several structural and statistical properties of modern genes: a repeated AUG codon appears spontaneously (and is thus made available for becoming a start signal), the form AUG/STOP emerges, and frequency patterns resemble those of present genes. The whole set of rings was also compared to a database of tRNAs, considering the conserved positions (located in the free parts of the molecule, essentially the loops); the ring that most closely matched tRNA sequences-and matched, in fact, the consensus of tRNA at all the aligned positions-was AL, the same ring independently selected before. The unselected emergence of gene-like features through two simple selection steps and the close similarity between the finally selected ring and tRNA (including some remarkable features of the resulting alignment) suggest a possible link between the prebiotic world and the first biological molecules, which is amenable for experimental testing. Even if our scenario is partially wrong, the unlikely coincidences should provide useful hints for other efforts.     

Effects of N2,N2-dimethylguanosine on RNA structure and stability: crystal structure of an RNA duplex with tandem m2 2G:A pairs

Methylation of the exocyclic amino group of guanine is a relatively common modification in rRNA and tRNA. Single methylation (N²-methylguanosine, m²G) is the second most frequently encountered nucleoside analog in Escherichia coli rRNAs. The most prominent case of dual methylation (N²,N²-dimethylguanosine, m²₂G) is found in the majority of eukaryotic tRNAs at base pair m²₂G26:A44. The latter modification eliminates the ability of the N² function to donate in hydrogen bonds and alters its pairing behavior, notably vis-à-vis C. Perhaps a less obvious consequence of the N²,N²-dimethyl modification is its role in controlling the pairing modes between G and A. We have determined the crystal structure of a 13-mer RNA duplex with central tandem m²₂G:A pairs. In the structure both pairs adopt an imino-hydrogen bonded, pseudo-Watson–Crick conformation. Thus, the sheared conformation frequently seen in tandem G:A pairs is avoided due to a potential steric clash between an N²-methyl group and the major groove edge of A. Additionally, for a series of G:A containing self-complementary RNAs we investigated how methylation affects competitive hairpin versus duplex formation based on UV melting profile analysis.     

An Extended RNA Code and its Relationship to the Standard Genetic Code: An Algebraic and Geometrical Approach

An algebraic and geometrical approach is used to describe the primaeval RNA code and a proposed Extended RNA code. The former consists of all codons of the type RNY, where R means purines, Y pyrimidines, and N any of them. The latter comprises the 16 codons of the type RNY plus codons obtained by considering the RNA code but in the second (NYR type), and the third, (YRN type) reading frames. In each of these reading frames, there are 16 triplets that altogether complete a set of 48 triplets, which specify 17 out of the 20 amino acids, including AUG, the start codon, and the three known stop codons. The other 16 codons, do not pertain to the Extended RNA code and, constitute the union of the triplets YYY and RRR that we define as the RNA-less code. The codons in each of the three subsets of the Extended RNA code are represented by a four-dimensional hypercube and the set of codons of the RNA-less code is portrayed as a four-dimensional hyperprism. Remarkably, the union of these four symmetrical pairwise disjoint sets comprises precisely the already known six-dimensional hypercube of the Standard Genetic Code (SGC) of 64 triplets. These results suggest a plausible evolutionary path from which the primaeval RNA code could have originated the SGC, via the Extended RNA code plus the RNA-less code. We argue that the life forms that probably obeyed the Extended RNA code were intermediate between the ribo-organisms of the RNA World and the last common ancestor (LCA) of the Prokaryotes, Archaea, and Eucarya, that is, the cenancestor. A general encoding function, E, which maps each codon to its corresponding amino acid or the stop signal is also derived. In 45 out of the 64 cases, this function takes the form of a linear transformation F, which projects the whole six-dimensional hypercube onto a four-dimensional hyperface conformed by all triplets that end in cytosine. In the remaining 19 cases the function E adopts the form of an affine transformation, i.e., the composition of F with a particular translation. Graphical representations of the four local encoding functions and E, are illustrated and discussed. For every amino acid and for the stop signal, a single triplet, among those that specify it, is selected as a canonical representative. From this mapping a graphical representation of the 20 amino acids and the stop signal is also derived. We conclude that the general encoding function E represents the SGC itself.     

鹅膏毒肽与RNA聚合酶II相互作用的分子机制研究

研究鹅膏毒肽与RNA聚合酶II相互作用的分子机制,利用分子对接方法获得了9种鹅膏毒肽与RNA聚合酶II相互作用的结合模式、结合位点、对接能和抑制常数等信息,并对鹅膏毒肽的毒性与结构间的构效关系进行了考察。结果表明：利用分子对接方法获得的鹅膏毒肽与RNA聚合酶II相互作用的信息与实验结果相一致;不同R2取代基引起毒素与聚合酶II结合能力强弱不同,从而导致鹅膏毒肽分子间的毒性差异。结果证实了运用分子对接方法探索多肽分子与蛋白质相互作用的可行性,为在分子水平上研究多肽与蛋白质的相互作用开拓了新的思路。     

Structure of the genetic code suggested by the hydropathy correlation between anticodons and amino acid residues

The correlation between hydropathies of anticodons and amino acids, detected by other authors utilizing scales of amino acid molecules in solution, was improved with the utilization of scales of amino acid residues in proteins. Three partitions were discerned in the correlation plot with the principal dinucleotides of anticodons (pDiN, excluding the wobble position). (a) The set of outliers of the correlation: Gly-CC, Pro-GG, Ser-GA and Ser-CU. The amino acids are consistently small, hydro-apathetic, stabilizers of protein N-ends, preferred in aperiodic protein conformations and belong to synthetases class II. The pDiN sequences are representative of the homogeneous sector (triplets NRR and NYY), distinguished from the mixed sector (triplets NRY and NYR), that depict a 70% correspondence to the synthetases class II and I, respectively. The triplet pairs proposed to be responsible for the coherence in the set of outliers are of the palindromic kind, where the lateral bases are the same, CCC: GGG and AGA: UCU. This suggests that UCU previously belonged to Ser, adding to other indications that the attribution of Arg to YCU was due to an expansion of the Arg-tRNA synthetase specificity. The other attributions produced two correlation sets. (b) One corresponds to the remaining pDiN of the homogeneous sector, containing both synthetase classes; its regression line overlapped the one formed by the remaining attributions to class II. (c) The other contains the pDiN of the mixed sector and produced steeper slopes, especially with the class I attributions. It is suggested that the correlation was established when the amino acid composition of the protein synthetases became progressively enriched and that the set of outliers were the earliest to have been fixed.     

A hierarchical MS2/MS3 database search algorithm for automated analysis of phosphopeptide tandem mass spectra

A novel hierarchical MS²/MS³ database search algorithm has been developed to analyze MS²/MS³ phosphopeptides proteomic data. The algorithm is incorporated in an automated database search program, MassMatrix. The algorithm matches experimental MS² spectra against a supplied protein database to determine candidate peptide matches. It then matches the corresponding experimental MS³ spectra against those candidate peptide matches. The MS² and MS³ spectra are used in concert to arrive at peptide matches with overall higher confidence rather than combining MS² and MS³ data searched separately. Receiver operating characteristic analysis showed that hierarchical MS²/MS³ database searches with MassMatrix had better sensitivity and specificity than the two‐stage MS²/MS³ database searches obtained with MassMatrix, MASCOT, and X!Tandem. A greater number of true peptide matches at a given false rate were identified by use of this new algorithm for data collected on both LCQ and LTQ‐FTICR mass spectrometers. The additional MS³ spectral data also improved the overall reliability and the number of true positives (TPs) due to the fact that the TPs of the MS²/MS³ search results had higher scores than those of the MS².     

Development of the genetic code: Insights from a fungal codon reassignment

The high conservation of the genetic code and its fundamental role in genome decoding suggest that its evolution is highly restricted or even frozen. However, various prokaryotic and eukaryotic genetic code alterations, several alternative tRNA-dependent amino acid biosynthesis pathways, regulation of tRNA decoding by diverse nucleoside modifications and recent in vivo incorporation of non-natural amino acids into prokaryotic and eukaryotic proteins, show that the code evolves and is surprisingly flexible. The cellular mechanisms and the proteome buffering capacity that support such evolutionary processes remain unclear. Here we explore the hypothesis that codon misreading and reassignment played fundamental roles in the development of the genetic code and we show how a fungal codon reassignment is enlightening its evolution.     

Structural Constraints and Mutational Bias in the Evolutionary Restoration of a Severe Deletion in RNA Phage MS2

A 4-nucleotide (nt) deletion was made in the 36-nt-long intercistronic region separating the coat and replicase genes of the single-stranded RNA phage MS2. This region is the focus of several RNA structures conferring high fitness. One such element is the operator hairpin, which, in the course of infection, will bind a coat-protein dimer, thereby precluding further replicase synthesis and initiating encapsidation. Another structure is a long-distance base pairing (MJ) controlling replicase expression. The 4-nt deletion does not directly affect the operator hairpin but it disrupts the MJ pairing. Its main effect, however, is a frame shift in the overlapping lysis gene. This gene starts in the upstream coat gene, runs through the 36-nt-long intercistronic region, and ends in the downstream replicase cistron. Here we report and interpret the spectrum of solutions that emerges when the crippled phage is evolved. Four different solutions were obtained by sequencing 40 plaques. Three had cured the frame shift in the lysis gene by inserting one nt in the loop of the operator hairpin causing its inactivation. Yet these low-fitness revertants could further improve themselves when evolved. The inactivated operator was replaced by a substitute and thereafter these revertants found several ways to restore control over the replicase gene. To allow for the evolutionary enrichment of low-probability but high-fitness revertants, we passaged lysate samples before plating. Revertants obtained in this way also restored the frame shift, but not at the expense of the operator. By taking larger and larger lysates samples for such bulk evolution, ever higher-fitness and lower-frequency revertants surfaced. Only one made it back to wild type. As a rule, however, revertants moved further and further away from the wild-type sequence because restorative mutations are, in the majority of cases, selected for their capacity to improve the phenotype by optimizing one of several potential alternative RNA foldings that emerge as a result of the initial deletion. This illustrates the role of structural constraints which limit the path of subsequent restorative mutations. [Reviewing Editor: Dr. John Hulsenbeck]     

Patterns of codon usage in two ciliates that reassign the genetic code: Tetrahymena thermophila and Paramecium tetraurelia

We used the recently sequenced genomes of the ciliates Tetrahymena thermophila and Paramecium tetraurelia to analyze the codon usage patterns in both organisms; we have analyzed codon usage bias, Gln codon usage, GC content and the nucleotide contexts of initiation and termination codons in Tetrahymena and Paramecium. We also studied how these trends change along the length of the genes and in a subset of highly expressed genes. Our results corroborate some of the trends previously described in Tetrahymena, but also negate some specific observations. In both genomes we found a strong bias toward codons with low GC content; however, in highly expressed genes this bias is smaller and codons ending in GC tend to be more frequent. We also found that codon bias increases along gene segments and in highly expressed genes and that the context surrounding initiation and termination codons are always AT rich. Our results also suggest differences in the efficiency of translation of the reassigned stop codons between the two species and between the reassigned codons. Finally, we discuss some of the possible causes for such translational efficiency differences.     

Relaxation kinetics of the interaction between RNA and metal-intercalators: the Poly(A).Poly(U)/platinum-proflavine system

The interactions of Poly(A).Poly(U) with the cis-platinum derivative of proflavine [{PtCl(tmen)}(2){HNC(13)H(7)(NHCH(2)CH(2))(2)}](+) (PRPt) and proflavine (PR) are investigated by spectrophotometry, spectrofluorimetry and T-jump relaxation at I=0.2M, pH 7.0, and T=25 degrees C. Base-dye interactions prevail at high RNA/dye ratio and binding isotherms analysis reveals that both dyes bind to Poly(A).Poly(U) according to the excluded site model (n=2). Only one relaxation effect is observed for the Poly(A).Poly(U)/PRPt system, whereas two effects are observed with Poly(A).Poly(U)/PR. The results agree with the sequence D+S <==> D, S <==> DS(I) <==> DS(II), where D,S is an external complex, DS(I) is a partially intercalated species, and DS(II) is the fully intercalated complex. Formation of DS(II) could be observed in the case of proflavine only. This result is interpreted by assuming that the platinum-containing residue of PRPt hinders the full intercalation of the acridine residue.