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A recent crystal structure of a ribosome complex undergoing partial translocation in the absence of elongation factor EF-G showed disruption of codon-anticodon pairing and slippage of the reading frame by -1, directly implicating EF-G in preservation of the translational reading frame. Among mutations identified in a random screen for dominant-lethal mutations of EF-G were a cluster of six that map to the tip of domain IV, which has been shown to contact the codon-anticodon duplex in trapped translocation intermediates. In vitro synthesis of a full-length protein using these mutant EF-Gs revealed dramatically increased -1 frameshifting, providing new evidence for a role for domain IV of EF-G in maintaining the reading frame. These mutations also caused decreased rates of mRNA translocation and rotational movement of the head and body domains of the 30S ribosomal subunit during translocation. Our results are in general agreement with recent findings from Rodnina and coworkers based on in vitro translation of an oligopeptide using EF-Gs containing mutations at two positions in domain IV, who found an inverse correlation between the degree of frameshifting and rates of translocation. Four of our six mutations are substitutions at positions that interact with the translocating tRNA, in each case contacting the RNA backbone of the anticodon loop. We suggest that EF-G helps to preserve the translational reading frame by preventing uncoupled movement of the tRNA through these contacts; a further possibility is that these interactions may stabilize a conformation of the anticodon that favors base-pairing with its codon., (© 2021 Niblett et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

A stop or nonsense codon is an in-frame triplet within a messenger RNA that signals the termination of translation. One common feature shared among all three nonsense codons (UAA, UAG, and UGA) is a uridine present at the first codon position. It has been recently shown that the conversion of this uridine into pseudouridine (Ψ) suppresses translation termination, both in vitro and in vivo. Furthermore, decoding of the pseudouridylated nonsense codons is accompanied by the incorporation of two specific amino acids in a nonsense codon-dependent fashion. Ψ differs from uridine by a single N¹H group at the C5 position; how Ψ suppresses termination and, more importantly, enables selective decoding is poorly understood. Here, we provide molecular rationales for how pseudouridylated stop codons are selectively decoded. Our analysis applies crystal structures of ribosomes in varying states of translation to consider weakened interaction of Ψ with release factor; thermodynamic and geometric considerations of the codon-anticodon base pairs to rank and to eliminate mRNA-tRNA pairs; the mechanism of fidelity check of the codon-anticodon pairing by the ribosome to evaluate noncanonical codon-anticodon base pairs and the role of water. We also consider certain tRNA modifications that interfere with the Ψ-coordinated water in the major groove of the codon-anticodon mini-helix. Our analysis of nonsense codons enables prediction of potential decoding properties for Ψ-modified sense codons, such as decoding ΨUU potentially as Cys and Tyr. Our results provide molecular rationale for the remarkable dynamics of ribosome decoding and insights on possible reprogramming of the genetic code using mRNA modifications.

In the universal genetic code, most amino acids can be encoded by multiple trinucleotide codons, and the choice among available codons can influence position-specific translation elongation rates. By using sequence-based ribosome profiling, we obtained transcriptome-wide profiles of in vivo ribosome occupancy as a function of codon identity in Caenorhabditis elegans and human cells. Particularly striking in these profiles was a universal trend of higher ribosome occupancy for codons translated via G:U wobble base-pairing compared with synonymous codons that pair with the same tRNA family using G:C base-pairing. These data support a model in which ribosomal translocation is slowed at wobble codon positions.

Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m⁵C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m⁵C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m⁵C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes' respective specificities for nucleotides C1404 and C1407.

The pandemic of 1918 was caused by an H1N1 influenza A virus, which is a negative strand RNA virus; however, little is known about the nature of its direct ancestral strains. Here we applied a broad genetic and phylogenetic analysis of a wide range of influenza virus genes, in particular the PB1 gene, to gain information about the phylogenetic relatedness of the 1918 H1N1 virus. We compared the RNA genome of the 1918 strain to many other influenza strains of different origin by several means, including relative synonymous codon usage (RSCU), effective number of codons (ENC), and phylogenetic relationship. We found that the PB1 gene of the 1918 pandemic virus had ENC values similar to the H1N1 classical swine and human viruses, but different ENC values from avian as well as H2N2 and H3N2 human viruses. Also, according to the RSCU of the PB1 gene, the 1918 virus grouped with all human isolates and "classical" swine H1N1 viruses. The phylogenetic studies of all eight RNA gene segments of influenza A viruses may indicate that the 1918 pandemic strain originated from a H1N1 swine virus, which itself might be derived from a H1N1 avian precursor, which was separated from the bulk of other avian viruses in toto a long time ago. The high stability of the RSCU pattern of the PB1 gene indicated that the integrity of RNA structure is more important for influenza virus evolution than previously thought.

The choice of synonymous codons used to encode a polypeptide contributes to substantial differences in translation efficiency between genes. However, both the magnitude and the mechanisms of codon-mediated effects are unknown, as neither the effects of individual codons nor the parameters that modulate codon-mediated regulation are understood, particularly in eukaryotes. To explore this problem in Saccharomyces cerevisiae, we performed the first systematic analysis of codon effects on expression. We find that the arginine codon CGA is strongly inhibitory, resulting in progressively and sharply reduced expression with increased CGA codon dosage. CGA-mediated inhibition of expression is primarily due to wobble decoding of CGA, since it is nearly completely suppressed by coexpression of an exact match anticodon-mutated tRNA(Arg(UCG)), and is associated with generation of a smaller RNA fragment, likely due to endonucleolytic cleavage at a stalled ribosome. Moreover, CGA codon pairs are more effective inhibitors of expression than individual CGA codons. These results directly implicate decoding by the ribosome and interactions at neighboring sites within the ribosome as mediators of codon-specific translation efficiency.

Faithful decoding of the genetic information by the ribosome relies on kinetically driven mechanisms that promote selection of cognate substrates during elongation. Recently, we have shown that in addition to these kinetically driven mechanisms, the ribosome possesses a post peptidyl transfer quality control system that retrospectively monitors the codon-anticodon interaction in the P site, triggering substantial losses in the specificity of the A site during subsequent tRNA and RF selection when a mistake has occurred. Here, we report a detailed kinetic analysis of tRNA selection in the context of a mismatched P-site codon:anticodon interaction. We observe pleiotropic effects of a P-site mismatch on tRNA selection, such that near-cognate tRNA is processed by the ribosome almost as efficiently as cognate. In particular, after a miscoding event, near-cognate codon-anticodon complexes are stabilized on the ribosome to an extent similar to that observed for cognate ones. Moreover, the two observed forward rates of GTPase activation and accommodation are greatly accelerated (∼10-fold) for near-cognate tRNAs. Because the ensemble of effects of a mismatched P site on substrate selection were found to be different from those reported for other ribosomal perturbations and miscoding agents, we propose that the structural integrity of the mRNA-tRNA helix in the P site provides a distinct molecular switch that dictates the specificity of the A site.

The selection of cognate tRNAs during translation is specified by a kinetic discrimination mechanism driven by distinct structural states of the ribosome. While the biochemical steps that drive the tRNA selection process have been carefully documented, it remains unclear how recognition of matched codon:anticodon helices in the small subunit facilitate global rearrangements in the ribosome complex that efficiently promote tRNA decoding. Here we use an in vitro selection approach to isolate tRNA(Trp) miscoding variants that exhibit a globally perturbed tRNA tertiary structure. Interestingly, the most substantial distortions are positioned in the elbow region of the tRNA that closely approaches helix 69 (H69) of the large ribosomal subunit. The importance of these specific interactions to tRNA selection is underscored by our kinetic analysis of both tRNA and rRNA variants that perturb the integrity of this interaction.

Analysis of 100 complete sets of the cytoplasmic elongator tRNA genes from Bacteria, Archaea, and Eukarya pointed to correspondences between types of anticodon and composition of the rest of the tRNA body. The number of the hydrogen bonds formed between the complementary nucleotides in the anticodon-codon duplex appeared as a major quantitative parameter determining covariations in all three domains of life. Our analysis has supported and advanced the "extended anticodon" concept that is based on the argument that the decoding performance of the anticodon is enhanced by selection of a matching anticodon stem-loop sequence, as reported by Yarus in 1982. In addition to the anticodon stem-loop, we have found covariations between the anticodon nucleotides and the composition of the distant regions of their respective tRNAs that include dihydrouridine (D) and thymidyl (T) stem-loops. The majority of the covariable tRNA positions were found at the regions with the increased dynamic potential--such as stem-loop and stem-stem junctions. The consistent occurrences of the covariations on the multigenomic level suggest that the number and pattern of the hydrogen bonds in the anticodon-codon duplex constitute a major factor in the course of translation that is reflected in the fine-tuning of the tRNA composition and structure.

Translating ribosomes can shift reading frame at specific sites with high efficiency for gene expression purposes. The most common type of shift to the -1 frame involves a tandem realignment of two anticodons from pairing with mRNA sequence of the form X XXY YYZ to XXX YYY Z where the spaces indicate the reading frame. The predominant -1 shift site of this type in eubacteria is A AAA AAG. The present work shows that in Escherichia coli the identity of the 6 nt 3' of this sequence can be responsible for a 14-fold variation in frameshift frequency. The first 3' nucleotide has the primary effect, with, in order of decreasing efficiency, U > C > A > G. This effect is independent of other stimulators of frameshifting. It is detected with other X XXA AAG sequences, but not with several other heptameric -1 shift sites. Pairing of E. coli tRNALYS with AAG is especially weak at the third codon position. We propose that strong stacking of purines 3' of AAG stabilizes pairing of tRNALys, diminishing the chance of codon:anticodon dissociation that is a prerequisite for the realignment involved in frameshifting.

The 18S rRNA environment of the mRNA at the decoding site of human 80S ribosomes has been studied by cross-linking with derivatives of hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group either at the N7 atom of the guanine or at the C5 atom of the 5'-terminal uracil residue. The location of the codons on the ribosome at A, P, or E sites has been adjusted by the cognate tRNAs. Three types of complexes have been obtained for each type derivative, namely, (1) codon UUU and Phe-tRNAPhe at the P site (codon GUU at the A site), (2) codon UUU and tRNAPhe at the P site and PheVal-tRNAVal at the A site, and (3) codon GUU and Val-tRNAVal at the P site (codon UUU at the E site). This allowed the placement of modified nucleotides of the mRNA analog at positions -3, +1, or +4 on the ribosome. Mild UV irradiation resulted in tRNA-dependent crosslinking of the mRNA analogs to the 18S rRNA. Nucleotide G961 crosslinked to mRNA position -3, nucleotide G1207 to position +1, and A1823 together with A1824 to position +4. All of these nucleotides are located in the most strongly conserved regions of the small subunit RNA structure, and correspond to nucleotides G693, G926, G1491, and A1492 of bacterial 16S rRNA. Three of them (with the exception of G1491) had been found earlier at the 70S ribosomal decoding site. The similarities and differences between the 16S and 18S rRNA decoding sites are discussed.

The methylation of 2'-hydroxyl groups is one of the most common posttranscriptional modifications of naturally occurring stable RNA molecules. Some tRNA species have a 2'-O-methyl nucleoside at the first position of the anticodon, and it was suggested that this modification stabilizes the codon-anticodon duplex. However, no tRNA species have been found to have the modification at the second or third position of the anticodon. In the present study, we measured the effects of anticodon 2'-O-methylation on the codon-reading efficiencies of the anticodon variants of the unmodified forms of Escherichia coli tRNA1(Ser), using a cell-free protein synthesis assay. The modification of C in the first position of the anticodon into 2'-O-methylcytidine increased the efficiency of reading the G-ending codon. On the other hand, the modifications of the second and/or third positions were detrimental to the codon-reading activity. Thus, 2'-hydroxyl groups at the second and third positions of the anticodon may have some role in the translation reaction, and this may be the reason why 2'-O-methyl nucleosides are not found in these positions within natural tRNA species.

In the genetic code, triplet codons and amino acids can be shown to be related by chemical principles. Such chemical regularities could be created either during the code's origin or during later evolution. One such chemical principle can now be shown experimentally. Natural or particularly selected RNA binding sites for at least three disparate amino acids (arginine, isoleucine, and tyrosine) are enriched in codons for the cognate amino acid. Currently, in 517 total nucleotides, binding sites contain 2.4-fold more codon sequences than surrounding nucleotides. The aggregate probability of this enrichment is 10(-7) to 10(-8), had codons and binding site sequences been independent. Thus, at least some primordial coding assignments appear to have exploited triplets from amino acid binding sites as codons.

If the genetic code arose in an RNA world, present codon assignments may reflect primordial RNA-amino acid affinities. Whether aptamers selected from random pools to bind free amino acids do so using the cognate codons at their binding sites has been controversial. Here we defend and extend our previous analysis of arginine binding sites, and propose a model for the maintenance of codon-amino acid interactions through the evolution of amino acids from ribozyme cofactors into the building blocks of proteins.

It has been suggested that in vitro selection experiments can provide information not only on what might have occurred during the evolution of the RNA world, but can in fact yield insights into particular features of the RNA world. In particular, it has been suggested that the sequences of anti-amino acid aptamers can provide clues to the origin of the genetic code, and that there is a statistically significant association between motifs found in aptamers and codons. We argue that the suggested connections between modern motifs and ancient sequences are logically tenuous, and show that there is no statistically meaningful association between motifs found in aptamers and codons.