Near-cognate aminoacyl-tRNAs Research Articles

Single-Molecule Studies of Cognate and Near-Cognate Elongation in an in vitro Eukaryotic Translation System.

The ribosome plays a central role in translation of the genetic code into amino acid sequences during synthesis of polypeptides. During each cycle of peptide elongation, the ribosome must discriminate between correct and incorrect aminoacyl-tRNAs according to the codon present in its A-site. Ribosomes rely on a complex sequence of proofreading mechanisms to minimize erroneous selection of incorrect aminoacyl-tRNAs that would lead to mistakes in translation. These mechanisms have been studied extensively in prokaryotic organisms, but eukaryotic elongation is less well understood. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) with an in vitro eukaryotic translation system to investigate tRNA selection and subsequent steps during peptide elongation. We compared accommodation of a tryptophan-aminoacyl-tRNA into the ribosomal A-site containing either a cognate or near-cognate codon and unexpectedly found that, following an initial slow sampling event, subsequent near-cognate sampling events proceeded more rapidly than the initial event. Further, we found a strong negative correlation between the concentration of near-cognate aminoacyl-tRNA and the efficiency of tRNA accommodation. These novel characteristics of near-cognate interaction with the eukaryotic ribosome suggest that rejection of a near-cognate tRNAs leads to formation of an altered ribosomal conformation that assists in rejecting subsequent incorrect tRNA interactions.

Error-prone protein synthesis recapitulates early symptoms of Alzheimer disease in aging mice.

Age-related neurodegenerative diseases (NDDs) are associated with the aggregation and propagation of specific pathogenic protein species (e.g., Aβ, α-synuclein). However, whether disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation, with its frequent outcome of random protein misfolding, is sufficient to recapitulate many early features of NDDs, including perturbed Ca2+ signaling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in behavioral changes reminiscent of early Alzheimer disease (AD), such as learning and memory deficits, maladaptive emotional responses, epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation, accompanied by hippocampal NPY expression and cerebral glucose hypometabolism. Collectively, our findings suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs, providing an alternative framework for understanding the initiation of AD.

Ribosome selectivity and nascent chain context in modulating the incorporation of fluorescent non-canonical amino acid into proteins

Fluorescence reporter groups are important tools to study the structure and dynamics of proteins. Genetic code reprogramming allows for cotranslational incorporation of non-canonical amino acids at any desired position. However, cotranslational incorporation of bulky fluorescence reporter groups is technically challenging and usually inefficient. Here we analyze the bottlenecks for the cotranslational incorporation of NBD-, BodipyFL- and Atto520-labeled Cys-tRNACys into a model protein using a reconstituted in-vitro translation system. We show that the modified Cys-tRNACys can be rejected during decoding due to the reduced ribosome selectivity for the modified aa-tRNA and the competition with native near-cognate aminoacyl-tRNAs. Accommodation of the modified Cys-tRNACys in the A site of the ribosome is also impaired, but can be rescued by one or several Gly residues at the positions −1 to −4 upstream of the incorporation site. The incorporation yield depends on the steric properties of the downstream residue and decreases with the distance from the protein N-terminus to the incorporation site. In addition to the full-length translation product, we find protein fragments corresponding to the truncated N-terminal peptide and the C-terminal fragment starting with a fluorescence-labeled Cys arising from a StopGo-like event due to a defect in peptide bond formation. The results are important for understanding the reasons for inefficient cotranslational protein labeling with bulky reporter groups and for designing new approaches to improve the yield of fluorescence-labeled protein.

Drug Repurposing Patent Applications April–June 2021

Drug Repurposing Patent Applications April–June 2021

Insights into the base-pairing preferences of 8-oxoguanosine on the ribosome.

Of the four bases, guanine is the most susceptible to oxidation, which results in the formation of 8-oxoguanine (8-oxoG). In protein-free DNA, 8-oxodG adopts the syn conformation more frequently than the anti one. In the syn conformation, 8-oxodG base pairs with dA. The equilibrium between the anti and syn conformations of the adduct are known to be altered by the enzyme recognizing 8-oxodG. We previously showed that 8-oxoG in mRNA severely disrupts tRNA selection, but the underlying mechanism for these effects was not addressed. Here, we use miscoding antibiotics and ribosome mutants to probe how 8-oxoG interacts with the tRNA anticodon in the decoding center. Addition of antibiotics and introduction of error-inducing mutations partially suppressed the effects of 8-oxoG. Under these conditions, rates and/or endpoints of peptide-bond formation for the cognate (8-oxoG•C) and near-cognate (8-oxoG•A) aminoacyl-tRNAs increased. In contrast, the antibiotics had little effect on other mismatches, suggesting that the lesion restricts the nucleotide from forming other interactions. Our findings suggest that 8-oxoG predominantly adopts the syn conformation in the A site. However, its ability to base pair with adenosine in this conformation is not sufficient to promote the necessary structural changes for tRNA selection to proceed.

Identification of the amino acids inserted during suppression of CFTR nonsense mutations and determination of their functional consequences.

In-frame premature termination codons (PTCs) account for ∼11% of all disease-associated mutations. PTC suppression therapy utilizes small molecules that suppress translation termination at a PTC to restore synthesis of a full-length protein. PTC suppression is mediated by the base pairing of a near-cognate aminoacyl-tRNA with a PTC and subsequently, the amino acid becomes incorporated into the nascent polypeptide at the site of the PTC. However, little is known about the identity of the amino acid(s) inserted at a PTC during this process in mammalian cells, or how the surrounding sequence context influences amino acid incorporation. Here, we determined the amino acids inserted at the cystic fibrosis transmembrane conductance regulator (CFTR) W1282X PTC (a UGA codon) in the context of its three upstream and downstream CFTR codons during G418-mediated suppression. We found that leucine, cysteine and tryptophan are inserted during W1282X suppression. Interestingly, these amino acids (and their proportions) are significantly different from those recently identified following G418-mediated suppression of the CFTR G542X UGA mutation. These results demonstrate for the first time that local mRNA sequence context plays a key role in near-cognate aminoacyl-tRNA selection during PTC suppression. We also found that some variant CFTR proteins generated by PTC suppression exhibit reduced maturation and activity, indicating the complexity of nonsense suppression therapy. However, both a CFTR corrector and potentiator enhanced activity of protein variants generated by G418-mediated suppression. These results suggest that PTC suppression in combination with CFTR modulators may be beneficial for the treatment of CF patients with PTCs.

Ensemble cryo-EM elucidates the mechanism of translation fidelity.

SUMMARYFaithful gene translation depends on accurate decoding, whose structural mechanism remains a matter of debate. Ribosomes decode mRNA codons by selecting cognate aminoacyl-tRNAs delivered by EF-Tu. We present high-resolution structural ensembles of ribosomes with cognate or near-cognate aminoacyl-tRNAs delivered by EF-Tu. Both cognate and near-cognate tRNA anticodons explore the A site of an open 30S subunit, while inactive EF-Tu is separated from the 50S subunit. A transient conformation of decoding-center nucleotide G530 stabilizes the cognate codon-anticodon helix, initiating step-wise “latching” of the decoding center. The resulting 30S domain closure docks EF-Tu at the sarcin-ricin loop of the 50S subunit, activating EF-Tu for GTP hydrolysis and ensuing aminoacyl-tRNA accommodation. By contrast, near-cognate complexes fail to induce the G530 latch, thus favoring open 30S pre-accommodation intermediates with inactive EF-Tu. This work unveils long-sought structural differences between the pre-accommodation of cognate and near-cognate tRNA that elucidate the mechanism of accurate decoding.

O6-Methylguanosine leads to position-dependent effects on ribosome speed and fidelity.

Nucleic acids are under constant assault from endogenous and environmental agents that alter their physical and chemical properties. O6-methylation of guanosine (m6G) is particularly notable for its high mutagenicity, pairing with T, during DNA replication. Yet, while m6G accumulates in both DNA and RNA, little is known about its effects on RNA. Here, we investigate the effects of m6G on the decoding process, using a reconstituted bacterial translation system. m6G at the first and third position of the codon decreases the accuracy of tRNA selection. The ribosome readily incorporates near-cognate aminoacyl-tRNAs (aa-tRNAs) by forming m6G-uridine codon–anticodon pairs. Surprisingly, the introduction of m6G to the second position of the codon does not promote miscoding, but instead slows the observed rates of peptide-bond formation by >1000-fold for cognate aa-tRNAs without altering the rates for near-cognate aa-tRNAs. These in vitro observations were recapitulated in eukaryotic extracts and HEK293 cells. Interestingly, the analogous modification N6-methyladenosine (m6A) at the second position has only a minimal effect on tRNA selection, suggesting that the effects on tRNA selection seen with m6G are due to altered geometry of the base pair. Given that the m6G:U base pair is predicted to be nearly indistinguishable from a Watson-Crick base pair, our data suggest that the decoding center of the ribosome is extremely sensitive to changes at the second position. Our data, apart from highlighting the deleterious effects that these adducts pose to cellular fitness, shed new insight into decoding and the process by which the ribosome recognizes codon–anticodon pairs.

Class 1 CF Mutations

Since the discovery of the gene that causes Cystic Fibrosis, our knowledge of how mutations in this gene cause the varied pathophysiological manifestations of this disease has increased substantially. This knowledge has led to the possibility of new therapeutic approaches aimed at the basic defect. Class I mutations of CFTR include premature termination codons (PTCs) or stop codons. In the last 10 years there has been a concerted international effort to utilize the concept of read-through of the stop codon producing full length functioning CFTR protein. This author considers that this approach will result in clinical trials in CF patients carrying these mutations. Class I mutations include PTCs or nonsense codons. A nonsense mutation is a single point alteration in DNA that results in the inappropriate presence of a UAA, UAG, or UGA stop codon in the protein-coding region of the corresponding messenger RNA (mRNA) transcript. Such a stop codon causes premature cessation of translation, with protein truncation leading to loss of function and consequent disease. Nonsense mutations are responsible for about 10% of cystic fibrosis cases worldwide. However, in Israel, nonsense mutations are the cause of cystic fibrosis in most patients (Kerem et al., 1997). As such mutations produce little functional CFTR, these patients usually have a phenotype of CF with exocrine pancreatic insufficiency. The increased understanding of ribosomal function, the process of translation, and small molecules that change the interaction between the ribosome and mRNA have led to the identification of several agents that are capable of suppressing PTCs. This has resulted in a novel strategy to treat CF and other genetic disorders caused by PTCs by restoring full length protein. Aminoglycoside antibiotics were the first drugs demonstrated to suppress PTCs in disease-causing mutations, allowing the translation of full length proteins (Hermann, 2007). Aminoglycosides are antibacterial agents, their mode of action is interfering with normal translation via binding to the bacteria 16S rRNA. There is reduced discrimination between cognate and near-cognate tRNA hence reducing translational fidelity. Eventually, there is accumulation of truncated and non-functioning proteins resulting in bacterial cell death. Gorini and Kataja (1964) demonstrated that aminoglycosides may suppress PTCs and lead to full length translation in E. coli. Aminoglycosides may also bind to human 18S rRNA subunit reducing discrimination of near-cognate tRNAs. This interaction is less stable than in bacteria but may be sufficient to lead to an insertion of a near-cognate aminoacyl-tRNA into the ribosomal A site that is subsequently incorporated into the polypeptide chain. Howard et al. (1996) described PTC suppression by the synthetic aminoglycoside geneticin (G418) to restore function in HeLa cells expressing nonsense codons in 1996. This pivotal work was extended to four nonsense mutations of cftr who were expressed by the human airway cell line IB3-1.In this study, the commonly used aminoglycoside, gentamicin, was incubated with these cells and full length protein was produced (Bedwell et al., 1997).

Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

The molecular basis of the induced-fit mechanism that determines the fidelity of protein synthesis remains unclear. Here, we isolated mutations in 16S rRNA that increase the rate of miscoding and stop codon read-through. Many of the mutations clustered along interfaces between the 30S shoulder domain and other parts of the ribosome, strongly implicating shoulder movement in the induced-fit mechanism of decoding. The largest subset of mutations mapped to helices h8 and h14. These helices interact with each other and with the 50S subunit to form bridge B8. Previous cryo-EM studies revealed a contact between h14 and the switch 1 motif of EF-Tu, raising the possibility that h14 plays a direct role in GTPase activation. To investigate this possibility, we constructed both deletions and insertions in h14. While ribosomes harboring a 2-base-pair (bp) insertion in h14 were completely inactive in vivo, those containing a 2-bp deletion retained activity but were error prone. In vitro, the truncation of h14 accelerated GTP hydrolysis for EF-Tu bearing near-cognate aminoacyl-tRNA, an effect that can largely account for the observed miscoding in vivo. These data show that h14 does not help activate EF-Tu but instead negatively controls GTP hydrolysis by the factor. We propose that bridge B8 normally acts to counter inward rotation of the shoulder domain; hence, mutations in h8 and h14 that compromise this bridge decrease the stringency of aminoacyl-tRNA selection.

Resampling and Editing of Mischarged tRNA Prior to Translation Elongation

Faithful translation of the genetic code depends on the GTPase EF-Tu delivering correctly charged aminoacyl-tRNAs to the ribosome for pairing with cognate codons. The accurate coupling of cognate amino acids and tRNAs by the aminoacyl-tRNA synthetases is achieved through a combination of substrate specificity and product editing. Once released by aminoacyl-tRNA synthetases, both cognate and near-cognate aminoacyl-tRNAs were considered to be committed to ribosomal protein synthesis through their association with EF-Tu. Here we show instead that aminoacyl-tRNAs in ternary complex with EF-Tu*GTP can readily dissociate and rebind to aminoacyl-tRNA synthetases. For mischarged species, this allows resampling by the product editing pathway, leading to a reduction in the overall error rate of aminoacyl-tRNA synthesis. Resampling of mischarged tRNAs was shown to increase the accuracy of translation over ten fold during in vitro protein synthesis, supporting the presence of an additional quality control step prior to translation elongation.

Mutational Analysis of S12 Protein and Implications for the Accuracy of Decoding by the Ribosome

The fidelity of aminoacyl-tRNA selection by the ribosome depends on a conformational switch in the decoding center of the small ribosomal subunit induced by cognate but not by near-cognate aminoacyl-tRNA. The aminoglycosides paromomycin and streptomycin bind to the decoding center and induce related structural rearrangements that explain their observed effects on miscoding. Structural and biochemical studies have identified ribosomal protein S12 (as well as specific nucleotides in 16S ribosomal RNA) as a critical molecular contributor in distinguishing between cognate and near-cognate tRNA species as well as in promoting more global rearrangements in the small subunit, referred to as “closure.” Here we use a mutational approach to define contributions made by two highly conserved loops in S12 to the process of tRNA selection. Most S12 variant ribosomes tested display increased levels of fidelity (a “restrictive” phenotype). Interestingly, several variants, K42A and R53A, were substantially resistant to the miscoding effects of paromomycin. Further characterization of the compromised paromomycin response identified a probable second, fidelity-modulating binding site for paromomycin in the 16S ribosomal RNA that facilitates closure of the small subunit and compensates for defects associated with the S12 mutations.

Mutational analysis reveals two independent molecular requirements during transfer RNA selection on the ribosome

Accurate discrimination between cognate and near-cognate aminoacyl-tRNAs during translation relies on the specific acceleration of forward rate constants for cognate tRNAs. Such specific rate enhancement correlates with conformational changes in the tRNA and small ribosomal subunit that depend on an RNA-specific type of interaction, the A-minor motif, between universally conserved 16S ribosomal RNA nucleotides and the cognate codon-anticodon helix. We show that perturbations of these two components of the A-minor motif, the conserved rRNA bases and the codon-anticodon helix, result in distinct outcomes. Although both cause decreases in the rates of tRNA selection that are rescued by aminoglycoside antibiotics, only disruption of the codon-anticodon helix is overcome by a miscoding tRNA variant. On this basis, we propose that two independent molecular requirements must be met to allow tRNAs to proceed through the selection pathway, providing a mechanism for exquisite control of fidelity during this step in gene expression.

Fidelity in protein synthesis

Fidelity in protein synthesis

Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system.

The translation machinery recognizes codons that enter the ribosomal A site with remarkable accuracy to ensure that polypeptide synthesis proceeds with a minimum of errors. When a termination codon enters the A site of a eukaryotic ribosome, it is recognized by the release factor eRF1. It has been suggested that the recognition of translation termination signals in these organisms is not limited to a simple trinucleotide codon, but is instead recognized by an extended tetranucleotide termination signal comprised of the stop codon and the first nucleotide that follows. Interestingly, pharmacological agents such as aminoglycoside antibiotics can reduce the efficiency of translation termination by a mechanism that alters this ribosomal proofreading process. This leads to the misincorporation of an amino acid through the pairing of a near-cognate aminoacyl tRNA with the stop codon. To determine whether the sequence context surrounding a stop codon can influence aminoglycoside-mediated suppression of translation termination signals, we developed a series of readthrough constructs that contained different tetranucleotide termination signals, as well as differences in the three bases upstream and downstream of the stop codon. Our results demonstrate that the sequences surrounding a stop codon can play an important role in determining its susceptibility to suppression by aminoglycosides. Furthermore, these distal sequences were found to influence the level of suppression in remarkably distinct ways. These results suggest that the mRNA context influences the suppression of stop codons in response to subtle differences in the conformation of the ribosomal decoding site that result from aminoglycoside binding.

Fidelity of the eukaryotic codon-anticodon interaction: interference by aminoglycoside antibiotics.

A homologous in vitro method was developed from Tetrahymena for ribosomal A-site binding of aminoacyl-tRNA to poly(uridylic acid)-programmed ribosomes with very low error frequency. The reaction mixture pH was the crucial factor in the stable A-site association of aminoacyl-tRNA with high fidelity. At a pH greater than 7.1, endogenous activity translocated A-site-bound aminoacyl-tRNA to the P site. If translocation was allowed to occur, a near-cognate amino-acyl-tRNA, Leu-tRNA, could stably bind to the ribosome by translocation to the ribosomal P site. Near-cognate aminoacyl-tRNA did not stably bind to either site when translocation was blocked. Misreading antibiotics stimulated the stable association of near-cognate aminoacyl-tRNA to the ribosomal A site, thereby increasing the error frequency by several orders of magnitude. Ribosome binding of total aminoacyl-tRNA near equilibrium was not inhibited by misreading antibiotics; however, initial rate kinetics of the binding reaction were dramatically altered such that a 6-fold rate increase was observed with paromomycin or hygromycin B. The rate increase was evident with both cognate and near-cognate aminoacyl-tRNAs. Several antibiotics were tested for misreading potency by the ribosome binding method. We found gentamicin G418 greater than paromomycin greater than neomycin greater than hygromycin B greater than streptomycin in the potentiation of misreading. Tetracycline group antibiotics effectively inhibited A-site aminoacyl-tRNA binding without promoting misreading.