Genetic code degeneracy is established by the decoding center of the ribosome.

The degeneracy of the genetic code confers a wide array of properties to coding sequences. Yet, its origin is still unclear. A structural analysis has shown that the stability of the Watson-Crick base pair at the second position of the anticodon-codon interaction is a critical parameter controlling the extent of non-specific pairings accepted at the third position by the ribosome, a flexibility at the root of degeneracy. Based on recent cryo-EM analyses, the present work shows that residue A1493 of the decoding center provides a significant contribution to the stability of this base pair, revealing that the ribosome is directly involved in the establishment of degeneracy. Building on existing evolutionary models, we show the evidence that the early appearance of A1493 and A1492 established the basis of degeneracy when an elementary kinetic scheme of translation was prevailing. Logical considerations on the expansion of this kinetic scheme indicate that the acquisition of the peptidyl transferase center was the next major evolutionary step, while the induced-fit mechanism, that enables a sharp selection of the tRNAs, necessarily arose later when G530 was acquired by the decoding center.

Peptidyl transferase center decompaction and structural constraints during early protein elongation on the ribosome.

Peptide bond formation on the ribosome requires that aminoacyl-tRNAs and peptidyl-tRNAs are properly positioned on the A site and the P site of the peptidyl transferase center (PTC) so that nucleophilic attack can occur. Here we analyse some constraints associated with the induced-fit mechanism of the PTC, that promotes this positioning through a compaction around the aminoacyl ester orchestrated by U2506. The physical basis of PTC decompaction, that allows the elongated peptidyl-tRNA to free itself from that state and move to the P site of the PTC, is still unclear. From thermodynamics considerations and an analysis of published ribosome structures, the present work highlights the rational of this mechanism, in which the free-energy released by the new peptide bond is used to kick U2506 away from the reaction center. Furthermore, we show the evidence that decompaction is impaired when the nascent peptide is not yet anchored inside the exit tunnel, which may contribute to explain why the first rounds of elongation are inefficient, an issue that has attracted much interest for about two decades. Results in this field are examined in the light of the present analysis and a physico-chemical correlation in the genetic code, which suggest that elementary constraints associated with the size of the side-chain of the amino acids penalize early elongation events.

Accurate characterization of Escherichia coli tRNA modifications with a simple method of deep-sequencing library preparation.

In conventional RNA high-throughput sequencing, modified bases prevent a large fraction of tRNA transcripts to be converted into cDNA libraries. Recent proposals aiming at resolving this issue take advantage of the interference of base modifications with RT enzymes to detect and identify them by establishing signals from aborted cDNA transcripts. Because some modifications, such as methyl groups, do almost not allow RT bypassing, demethylation and highly processive RT enzymes have been used to overcome these obstacles. Working with Escherichia coli as a model system, we show that with a conventional (albeit still engineered) RT enzyme and key optimizations in library preparation, all RT-impairing modifications can be highlighted along the entire tRNA length without demethylation procedure. This is achieved by combining deep-sequencing samples, which allows to establish aborted transcription signal of higher accuracy and reproducibility, with the potential for differentiating tiny differences in the state of modification of all cellular tRNAs. In addition, our protocol provides estimates of the relative tRNA abundance.

New Method for Improving LC/Time-of-Flight Mass Spectrometry Detection Limits Using Simultaneous Ion Counting and Waveform Averaging.

Simultaneous ion counting and waveform averaging implemented on a field-programmable gate array compiled with a high-speed digitizer was applied to ultraperformance liquid chromatography-time-of-flight mass spectrometric analysis of sulfa drugs. Ion counting was carried out by a "Peak Detection" (PKD) function that works together with signal averaging (AVG). Sulfadimidine (SDD) and sulfadimethoxine (SDMX) were measured in human serum (HS) model sample matrix. By using simultaneous PKD and AVG acquisition, we observed a unified calibration curve for more than 3 orders of magnitude of sample amounts (0.010-100.0 pmol). The ion count rate for the "practical" sample amounts, such as less than 1 pmol, was below 30%, which is suitable for PKD-based ion counting for quantitative accuracy and excellent peak identification performance. Samples containing 200 fmol or less could not be identified from the AVG waveform. Adding HS treated with acetonitrile severely suppressed the SDMX ion to less than one-half (58.1%). However, a linear response was observed for chromatographic peak area for analytes calculated from PKD waveforms. Also, the mass-resolving power calculated from the peak on the PKD waveform was 24% better than the corresponding AVG waveform, which also improves performance for analyte identification.

Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions.

Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of downstream genes to maintain nutritional homeostasis. Here, we report cocrystal and cryo-EM structures of Geobacillus kaustophilus and Bacillus subtilis T-box-tRNA complexes, detailing their multivalent, exquisitely selective interactions. The T-box forms a U-shaped molecular vise that clamps the tRNA, captures its 3' end using an elaborate 'discriminator' structure, and interrogates its aminoacylation state using a steric filter fashioned from a wobble base pair. In the absence of aminoacylation, T-boxes clutch tRNAs and form a continuously stacked central spine, permitting transcriptional readthrough or translation initiation. A modeled aminoacyl disrupts tRNA-T-box stacking, severing the central spine and blocking gene expression. Our data establish a universal mechanism of amino acid sensing on tRNAs and gene regulation by T-box riboswitches and exemplify how higher-order RNA-RNA interactions achieve multivalency and specificity.

D Amino Acids Highlight the Catalytic Power of the Ribosome.

The possible mechanism(s) by which ribosomes make peptide bonds during protein synthesis have been explored for decades. Yet, there is no agreement on how the catalytic site, the peptidyl transferase center (PTC), promotes this reaction. Here, we discuss the results of recent investigations of translation with D amino acids that provide fresh insights into that longstanding question.

Induced fit of the peptidyl-transferase center of the ribosome and conformational freedom of the esterified amino acids.

The catalytic site of most enzymes can efficiently handle only one substrate. In contrast, the ribosome is capable of polymerizing at a similar rate at least 20 different kinds of amino acids from aminoacyl-tRNA carriers while using just one catalytic site, the peptidyl-transferase center (PTC). An induced-fit mechanism has been uncovered in the PTC, but a possible connection between this mechanism and the uniform handling of the substrates has not been investigated. We present an analysis of published ribosome structures supporting the hypothesis that the induced fit eliminates unreactive rotamers predominantly populated for some A-site aminoacyl esters before induction. We show that this hypothesis is fully consistent with the wealth of kinetic data obtained with these substrates. Our analysis reveals that induction constrains the amino acids into a reactive conformation in a side-chain independent manner. It allows us to highlight the rationale of the PTC structural organization, which confers to the ribosome the very unusual ability to handle large as well as small substrates.

Fundamental amino acid mass distributions and entropy costs in proteomes.

We examine whether the frequency of amino acids across an organism's proteome is primarily determined by optimization to function or other factors, such as the structure of the genetic code. Considering all available proteins together, we first point out that the frequency of an amino acid in a proteome negatively correlates with its mass, suggesting that the genome preserves a fundamental distribution ruled by simple energetics. Given the universality of such distributions, one can use outliers, cysteine and leucine, to identify amino acids that deviate from this simple rule for functional purposes and examine those functions. We quantify the strength of such selection as the entropic cost outliers pay to defy the mass-frequency relation. Codon degeneracy of an amino acid partially explains the correlation between mass and frequency: light amino acids being typically encoded by highly degenerate codon families, with the exception of arginine. While degeneracy may be a factor in hard wiring the relationship between mass and frequency in proteomes, it does not provide a complete explanation. By examining extremophiles, we are able to show that this law weakens with temperature, likely due to protein stability considerations, thus the environment is essential.

A universal RNA structural motif docking the elbow of tRNA in the ribosome, RNAse P and T-box leaders.

The structure and function of conserved motifs constituting the apex of Stem I in T-box mRNA leaders are investigated. We point out that this apex shares striking similarities with the L1 stalk (helices 76-78) of the ribosome. A sequence and structure analysis of both elements shows that, similarly to the head of the L1 stalk, the function of the apex of Stem I lies in the docking of tRNA through a stacking interaction with the conserved G19:C56 base pair platform. The inferred structure in the apex of Stem I consists of a module of two T-loops bound together head to tail, a module that is also present in the head of the L1 stalk, but went unnoticed. Supporting the analysis, we show that a highly conserved structure in RNAse P formerly described as the J11/12-J12/11 module, which is precisely known to bind the elbow of tRNA, constitutes a third instance of this T-loop module. A structural analysis explains why six nucleotides constituting the core of this module are highly invariant among all three types of RNA. Our finding that major RNA partners of tRNA bind the elbow with a same RNA structure suggests an explanation for the origin of the tRNA L-shape.

Kinetics of bulge bases in small RNAs and the effect of pressure on it.

Due to their self-catalytic properties, small RNAs with bulge bases are hypothesized to be primordial molecules which could form elementary translation systems. Using molecular dynamics simulations, we study the binding propensity of small RNAs by calculating the free energy barrier corresponding to the looped out conformations of bulge bases, which presumably act as the binding sites for ligands in these small RNAs. We find that base flipping kinetics can proceed at atmospheric pressure but with a very small propensity. Furthermore, the free energy barrier associated with base flipping depends on the stacking with neighboring bases. Next, we studied the base flipping kinetics with pressure. We find that the free energy associated with base looping out increases monotonically as the pressure is increased. Furthermore, we calculate the mean first-passage time of conformational looping out of the bulge base using the diffusion of reaction coordinate associated with the base flipping on the underlying free energy surface. We find that the mean first-passage time associated with bulge looping out increases slowly upon increasing pressures P up to 2000 atm but changes dramatically for P>2000 atm. Finally, we discuss our results in the light of the role of hydration shell of water around RNA. Our results are relevant for the RNA world hypothesis.

Parents' perceptions of postschool years for young adults with developmental disabilities.

This qualitative study investigated parents' perceptions of the various roles they played in their adult children's lives during the post-high school years. Individual face-to-face interviews were conducted with 9 families of young adults with developmental disabilities. Findings indicated that families perceived the complexity of their roles as balancing between advocating for their adult children's needs while promoting independence and self-determination. The roles parents assumed as their children entered into adult life were those of collaborators, decision makers, and program evaluators, role models, trainers, mentors and instructors, and systems change agents. Parents often felt they were the safety net for their children and the back-up plan for service agencies. Parents' quotes illustrated the complexity of the roles they played as their young adult children with developmental disabilities entered adulthood.

Emergence of a code in the polymerization of amino acids along RNA templates.

The origin of the genetic code in the context of an RNA world is a major problem in the field of biophysical chemistry. In this paper, we describe how the polymerization of amino acids along RNA templates can be affected by the properties of both molecules. Considering a system without enzymes, in which the tRNAs (the translation adaptors) are not loaded selectively with amino acids, we show that an elementary translation governed by a Michaelis-Menten type of kinetics can follow different polymerization regimes: random polymerization, homopolymerization and coded polymerization. The regime under which the system is running is set by the relative concentrations of the amino acids and the kinetic constants involved. We point out that the coding regime can naturally occur under prebiotic conditions. It generates partially coded proteins through a mechanism which is remarkably robust against non-specific interactions (mismatches) between the adaptors and the RNA template. Features of the genetic code support the existence of this early translation system.

Degeneracy of the genetic code and stability of the base pair at the second position of the anticodon.

With an analysis of the structural constraints of the anticodon-codon interaction within the decoding center of the ribosome, we show that the extent of degeneracy at the third position of the anticodon is determined by the level of stability of the base pair at the second position.

Efficiency of a self-aminoacylating ribozyme: effect of the length and base-composition of its 3' extension.

Variants of a previously described small self-aminoacylating ribozyme are tested in order to uncover the potentialities of a 3' extension responsible for the esterification. The base-composition and the length of this specific part of the ribozyme are investigated. Very short extensions can still reach the active site, reflecting the small persistence length of RNA. The yield of aminoacylation is particularly high for ribozymes with extensions made up of a poly-U, for which a maximum of efficiency is observed for a total length of about 10 nucleotides. A simple model describing the behavior of this region of the ribozyme can account for the data.

Error minimization explains the codon usage of highly expressed genes in Escherichia coli.

Different organisms use synonymous codons with different preferences. Several measures have been introduced to compute the extent of codon usage bias within a gene or genome, among which the codon adaptation index (CAI) has been shown to be well correlated with mRNA levels of Escherichia coli. In this work an error adaptation index (eAI) is introduced, which estimates the level at which a gene can tolerate the effects of mistranslations. It is shown that the eAI has a strong correlation with CAI, as well as with mRNA levels, which suggests that the codons of highly expressed genes are selected so that mistranslation would have the minimum possible effect on the structure and function of the related proteins.

Folding of small RNAs displaying the GNC base-pattern: implications for the self-organization of the genetic system.

Theoretical arguments and statistical analyses of present-day coding sequences have long suggested the existence of primitive patterns in RNA sequences, which were thought to have been predominant at the time of the origin of the genetic code. The main propositions were centered around the base-patterns GNC and RNY, where R = A or G , Y = C or U and N = A, G, C or U. A theoretical model of the primitive process of translation explaining the origin of this type of pattern was recently published in the Journal of Theoretical Biology. On the basis of this previous analysis, and on physico-chemical evidence supporting the idea of the GNC base-pattern as the most primitive one, the present paper shows the results of folding simulations of small RNA strands displaying this pattern, which enabled us to specify the characteristics of the suggested primitive form of tRNA. This analysis is notably based on a conjecture of Eigen and Schuster of an early structural (or pattern) similarity between mRNA and tRNA, and, more specifically, of a "joint function of messenger and adaptor". Working with this conjecture, we show that the convergence of the primitive pool of RNAs toward a system containing a high proportion of sequences displaying the GNC base-pattern (according to the evolutionary model) is accompanied by a significant gain in stability of the translation process. In particular, it is demonstrated how the reading frame would be automatically discriminated without the presence of a start codon.

Amplification of the sequences displaying the pattern RNY in the RNA world: the translation --> translation/replication hypothesis.

Based on previous considerations published in J. theor. Biol., new analyses of the organization of the genetic system are reported in this paper. We show that theoretical considerations about the order observed in the genetic code table support the idea of a primitive self-aminoacylation process achieved by primordial tRNAs. The physico-chemical constraints connected with this process may explain why a primitive genetic system predominantly uses sequences with the codonic pattern RNN (R=purine; Y=pyrimidine; N=any of the four bases) to polymerize the amino acids into peptides through translation. These considerations lead us to propose the Translation --> Translation/Replication hypothesis, which may explain why only RNA sequences with the pattern RNY, instead of less restrictive RNN, are susceptible to amplification. Using these ideas, supported by properties of symmetry, features of the genetic code may be connected with the replication of specific RNA sequences in the RNA world.