AAA Codons Research Articles

Design and application of artificial rare L-lysine codons in Corynebacterium glutamicum.

Background: L-lysine is widely used in the feed, food, and pharmaceutical industries, and screening for high L-lysine-producing strains has become a key goal for the industry. Methods: We constructed the rare L-lysine codon AAA by corresponding tRNA promoter replacement in C. glutamicum. Additionally, a screening marker related to the intracellular L-lysine content was constructed by converting all L-lysine codons of enhanced green fluorescent protein (EGFP) into the artificial rare codon AAA. The artificial EGFP was then ligated into pEC-XK99E and transformed into competent Corynebacterium glutamicum 23604 cells with the rare L-lysine codon. After atmospheric and room-temperature plasma mutation and induction culture, 55 mutants (0.01% of total cells) with stronger fluorescence were sorted using flow cytometry, and further screened by fermentation in a 96-deep-well plate and 500mL shaker. Results: The fermentation results showed that the L-lysine production was increased by up to 9.7% in the mutant strains with higher fluorescence intensities, and that the highest screening positive rate was 69%, compared with that in the wild-type strain. Conclusion: The application of artificially constructed rare codons in this study represents an efficient, accurate, and simple method for screening other amino acid-producing microorganisms.

Characterization of zebrafish gp1ba mutant and modelling Bernard Soulier syndrome.

The aim of this study is to model classical Bernard Soulier Syndrome in the zebrafish by targeting Gp1ba. We obtained gp1ba mutant embryos from Zebrafish International Resource Center and grew them to adulthood. The tail clips from these fish were used to prepare DNA and sequenced to identify heterozygotes. They were then bred to obtain homozygotes. The mutation was confirmed by DNA sequencing as a termination codon UAA in place of AAA codon at position 886 in the gp1ba transcript. Thus, at the Pro-295, the Gp1ba protein could be terminated. The blood from gp1ba homozygous and heterozygous mutants showed decreased ristocetin-mediated agglutination in the whole blood agglutination assay. The gp1ba heterozygous and homozygous larvae were subjected to a laser-assisted arterial thrombosis assay, and the results showed the prolonged occlusion in the caudal artery. These results suggested that the gp1ba mutant had a bleeding phenotype. The blood smears from the adult gp1ba, heterozygous and homozygous mutants, showed macrothrombocytes, similar to the human GP1BA deficiency that showed giant platelets. The bleeding assay on these heterozygous and homozygous mutants showed greater bleeding than wildtype, confirming the above findings. Taken together, the characterization of gp1ba zebrafish mutant suggested an autosomal dominant mode of inheritance. The zebrafish gp1ba mutant models classical Bernard Soulier Syndrome and could be used for reversing this phenotype to identify novel factors by the genome-wide piggyback knockdown method.

Formula omitted]-circular codes

A message such as mRNA, which consists of continuous characters without separators (such as commas or spaces), can easily be decoded incorrectly if it is read in the wrong reading frame. One construct to theoretically avoid these reading frame errors is the class of block codes. However, the first hypothesis of Watson and Crick (1953) that block codes are used as a tool to avoid reading frame errors in coding sequences already failed because the four periodical codons AAA, CCC, GGG and UUU seem to play an important role in protein coding sequences. Even the class of circular codes later discovered by Arquès and Michel (1996) in coding sequences cannot contain a periodic codon. However, by incorporating the interpretation of the message into the robustness of the reading frame, the extension of circular codes to include periodic codons is theoretically possible. In this work, we introduce the new class of I-circular codes. Unlike circular codes, these codes allow frame shifts, but only if the decoded interpretation of the message is identical to the intended interpretation. In the following, the formal definition of I-circular codes is introduced and the maximum and the maximal size of I-circular codes are given based on the standard genetic code table. These numbers are calculated using a new graph-theoretic approach derived from the classical one for the class of circular codes. Furthermore, we show that all 216 maximum self-complementary C3-codes (see Fimmel et al., 2015) can be extended to larger I-circular codes. We present the increased code coverage of the 216 newly constructed I-circular codes based on the human coding sequences in chromosome 1. In the last section of this paper, we use the polarity of amino acids as an interpretation table to construct I-circular codes. In an optimization process, two maximum I-circular codes of length 30 are found.

The Sole Mycobacterium smegmatis MazF Toxin Targets tRNALys to Impart Highly Selective, Codon-Dependent Proteome Reprogramming.

Survival of mycobacteria, both free-living and host-dependent pathogenic species, is dependent on their ability to evade being killed by the stresses they routinely encounter. Toxin-antitoxin (TA) systems are unique to bacteria and archaea and are thought to function as stress survival proteins. Here, we study the activity of the endoribonuclease toxin derived from the MazEF TA system in Mycobacterium smegmatis, designated MazEF-ms. We first enlisted a specialized RNA-seq method, 5’ RNA-seq, to identify the primary RNA target(s) of the MazF-ms toxin. Just two tRNA species, tRNALys-UUU and tRNALys-CUU, were targeted for cleavage by MazF-ms at a single site within their anticodon sequence (UU↓U and CU↓U) to render these tRNAs nonfunctional for protein synthesis. The 5’ RNA-seq dataset also revealed hallmarks of ribosome stalling predominantly at Lys AAA codons even though both Lys tRNAs were cleaved by MazF-ms. Stalled ribosomes were then cleaved on their 5’ side by one or more RNases, resulting in very selective degradation of only those mRNAs harboring ribosomes stalled at Lys codons. This highly surgical, codon-dependent degradation of mRNA transcripts was validated using quantitative mass spectrometry of proteins that were newly synthesized during MazF-ms expression. The M. smegmatis proteome was altered as predicted, Lys AAA codon-rich proteins was downregulated while Lys AAA codon deficient proteins were upregulated. Analysis of specific subsets of proteins that were upregulated or downregulated was consistent with the growth-arrested phenotype of MazF-ms expressing cells. Curiously, the tRNA target and mechanism of action of MazF-ms paralleled that of one atypical MazF toxin in M. tuberculosis, suggesting manipulation of the levels of lysine tRNAs as the preferred conduit for reprogramming the proteomes via ribosome stalling at rare AAA codons in these GC-rich mycobacteria.

Coincidence detection stalls ribosome

Molecular Biology Translation of aberrant messenger RNAs that lack in-frame stop codons leads to malfunctioning proteins. To prevent this deleterious effect, cells have quality control processes to monitor translation into polyadenylated regions, including ribosome stalling. Using a combination of biochemistry and structural biology approaches, Chandrasekaran et al. show how ribosomes stall selectively on polyadenylated regions. Polylysine peptide (the codon AAA is decoded as lysine) in the exit channel of a ribosome slows translation, whereas a stabilized polyadenylated RNA helix in a ribosome decoding center blocks incoming transfer RNAs. Therefore, coincidence detection of both polylysine and polyadenylate allows polyadenylated regions, but not polylysine, within normal coding regions to halt translation. Nat. Struct. Mol. Biol. 26 , 1132 (2019).

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts.

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of invitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an invitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

Toxin-mediated ribosome stalling reprograms the Mycobacterium tuberculosis proteome

Mycobacterium tuberculosis readily adapts to survive a wide range of assaults by modifying its physiology and establishing a latent tuberculosis (TB) infection. Here we report a sophisticated mode of regulation by a tRNA-cleaving toxin that enlists highly selective ribosome stalling to recalibrate the transcriptome and remodel the proteome. This toxin, MazF-mt9, exclusively inactivates one isoacceptor tRNA, tRNALys43-UUU, through cleavage at a single site within its anticodon (UU↓U). Because wobble rules preclude compensation for loss of tRNALys43-UUU by the second M. tuberculosis lysine tRNA, tRNALys19-CUU, ribosome stalling occurs at in-frame cognate AAA Lys codons. Consequently, the transcripts harboring these stalled ribosomes are selectively cleaved by specific RNases, leading to their preferential deletion. This surgically altered transcriptome generates concomitant changes to the proteome, skewing synthesis of newly synthesized proteins away from those rich in AAA Lys codons toward those harboring few or no AAA codons. This toxin-mediated proteome reprogramming may work in tandem with other pathways to facilitate M. tuberculosis stress survival.

Titrating Gene Function in the Human Fungal Pathogen Candida albicans through Poly-Adenosine Tract Insertion.

A recent study demonstrated that the insertion of poly-adenosine (poly-A) tracts into an open reading frame can suppress expression of the encoded protein in both prokaryotic and eukaryotic species. Furthermore, the degree of suppression is proportional to the length of the poly-A insertion, which can therefore provide a reliable and predictable means to titrate a specific protein's expression. The goal of this study was to determine if this methodology can be applied to modulate the expression of proteins in the prevalent human fungal pathogen, Candida albicans Insertion of increasing numbers of AAA codons encoding lysine at the N terminus of the C. albicans lanosterol demethylase (Erg11p) progressively diminished expression without significantly reducing the levels of mRNA. This suggests that Erg11p expression was attenuated at the posttranscriptional level. A direct correlation between the number of AAA codons inserted and C. albicans susceptibility to the Erg11p inhibitor fluconazole was also noted, indicating a progressive loss of Erg11p activity. Finally, we constructed a series of C. albicans strains with 3 to 12 AAA codons inserted at the 5' end of the ARO1 gene, which encodes a pentafunctional enzyme catalyzing five sequential steps of the aromatic amino acid biosynthetic pathway. Increasing numbers of AAA codons progressively reduced the growth rate of C. albicans in standard laboratory medium, indicating a progressive loss of ARO biosynthetic activity. These data unequivocally demonstrate the potential utility of the poly-A insertion method to examine the phenotypic consequences of titrating target protein function in C. albicansIMPORTANCE Investigating a protein's functional importance at the whole-organism level usually involves altering its expression level or its specific activity and observing the consequences with respect to physiology or phenotype. Several approaches designed to partially or completely abolish the function of a gene, including its deletion from the genome and the use of systems that facilitate conditional expression, have been widely applied. However, each has significant limitations that are especially problematic in pathogenic microbes when it is desirable to determine if a particular gene is required for infection in an animal model. In this study, we sought to determine if an alternative approach-the insertion of poly-A repeats within the coding sequence of the gene-is sufficient to modulate its function in the prevalent human fungal pathogen C. albicans Our results confirm that this approach enables us to predictably and gradually titrate the expression level of a protein and thus to investigate the phenotypic consequences of various levels of gene/protein function.

Single-Frame, Multiple-Frame and Framing Motifs in Genes.

We study the distribution of new classes of motifs in genes, a research field that has not been investigated to date. A single-frame motif SF has no trinucleotide in reading frame (frame 0) that occurs in a shifted frame (frame 1 or 2), e.g., the dicodon AAACAA is as the trinucleotides AAA and CAA do not occur in a shifted frame. A motif which is not single-frame is multiple-frame . Several classes of motifs are defined and analysed. The distributions of single-frame motifs (associated with an unambiguous trinucleotide decoding in the two and directions) and 5′ unambiguous motifs (associated with an unambiguous trinucleotide decoding in the direction only) are analysed without and with constraints. The constraints studied are: initiation and stop codons, periodic codons , antiparallel complementarity and parallel complementarity. Taken together, these results suggest that the complementarity property involved in the antiparallel (DNA double helix, RNA stem) and parallel sequences could also be fundamental for coding genes with an unambiguous trinucleotide decoding in the two and directions or the direction only. Furthermore, the single-frame motifs with a property of trinucleotide decoding and the framing motifs (also called circular code motifs; first introduced by Michel (2012)) with a property of reading frame decoding may have been involved in the early life genes to build the modern genetic code and the extant genes. They could have been involved in the stage without anticodon-amino acid interactions or in the Implicated Site Nucleotides (ISN) of RNA interacting with the amino acids. Finally, the and dipeptides associated with the and dicodons, respectively, are studied and their importance for biology and the origin of life discussed.

Forces acting on codon bias in malaria parasites

Malaria parasite genomes have a range of codon biases, with Plasmodium falciparum one of the most AT-biased genomes known. We examined the make up of synonymous coding sites and stop codons in the core genomes of representative malaria parasites, showing first that local DNA context influences codon bias similarly across P. falciparum, P. vivax and P. berghei, with suppression of CpG dinucleotides and enhancement of CpC dinucleotides, both within and aross codons. Intense asexual phase gene expression in P. falciparum and P. berghei is associated with increased A3:G3 bias but reduced T3:C3 bias at 2-fold sites, consistent with adaptation of codons to tRNA pools and avoidance of wobble tRNA interactions that potentially slow down translation. In highly expressed genes, the A3:G3 ratio can exceed 30-fold while the T3:C3 ratio can be less than 1, according to the encoded amino acid and subsequent base. Lysine codons (AAA/G) show distinctive behaviour with substantially reduced A3:G3 bias in highly expressed genes, perhaps because of selection against frameshifting when the AAA codon is followed by another adenine. Intense expression is also associated with a strong bias towards TAA stop codons (found in 94% and 89% of highly expressed P. falciparum and P. berghei genes respectively) and a proportional rise in the TAAA stop ‘tetranucleotide’. The presence of these expression-linked effects in the relatively AT-rich malaria parasite species adds weight to the suggestion that AT-richness in the Plasmodium genus might be a fitness adaptation. Potential explanations for the relative lack of codon bias in P. vivax include the distinct features of its lifecycle and its effective population size over evolutionary time.

Preference of A/T ending codons in mitochondrial ATP6 gene under phylum Platyhelminthes: Codon usage of ATP6 gene in Platyhelminthes

Unequal usage of synonymous codons in the gene transcript for an amino acid is known as codon usage bias (CUB). It is a unique property of gene as well as genome. Mutation and natural selection are known to be the major factors that influence CUB. Other factors encompass gene expression level, GC content, codon position, recombination rate, RNA stability and gene length. CUB analysis helps in in-depth understanding of the molecular biology, genetics and genome evolution in a species. We used bioinformatic methods to explore the pattern of CUB in MT-ATP6 gene in different classes of platyhelminthes. The analysis is based on genetic code of translational table 14 of National Center of Biotechnology Information (NCBI) where the codon AAA codes for asparagine and TAA for tyrosine amino acid. The synonymous codon usage order (SCUO), an index of CUB, values in different classes namely cestoda, monogenea, rabditophora, trematoda and turbellaria of platyhelminths were found to be 0.43, 0.32, 0.49, 0.40 and 0.36, respectively which suggest that the codon usage bias of ATP6 gene was low (SCUO < 0.50). Highly significant correlation (p < 0.001) was found between SCUO and various GC contents indicating that GC composition had an influence on CUB. From the relative synonymous codon usage (RSCU) analysis on codons, we found most of the over-represented codons in all the classes were A/T ending types, which suggested that the preferred codons were influenced by compositional constraints. The PR2 plot revealed asymmetric usage of AT and GC bases among the four fold degenerate codon families with greater usage of G and T over A and C. Highly significant correlation (p < 0.001) was found between overall nucleotide composition and its 3rd codon position suggesting that both natural selection and mutation pressure might have influenced the CUB among different classes. Neutrality analysis revealed that natural selection might play a major role. Mutational responsive index (MRI) and translational selection (P2) values elucidated that selection for translational efficiency moderately affected the codon usage bias in MT-ATP6 gene.

Preferences of AAA/AAG codon recognition by modified nucleosides, τm5s2U34 and t6A37 present in tRNALys

Deficiency of 5-taurinomethyl-2-thiouridine, τm5s2U at the 34th ‘wobble’ position in tRNALys causes MERRF (Myoclonic Epilepsy with Ragged Red Fibers), a neuromuscular disease. This modified nucleoside of mt tRNALys, recognizes AAA/AAG codons during protein biosynthesis process. Its preference to identify cognate codons has not been studied at the atomic level. Hence, multiple MD simulations of various molecular models of anticodon stem loop (ASL) of mt tRNALys in presence and absence of τm5s2U34 and N6-threonylcarbamoyl adenosine (t6A37) along with AAA and AAG codons have been accomplished. Additional four MD simulations of multiple ASL mt tRNALys models in the context of ribosomal A-site residues have also been performed to investigate the role of A-site in recognition of AAA/AAG codons. MD simulation results show that, ASL models in presence of τm5s2U34 and t6A37 with codons AAA/AAG are more stable than the ASL lacking these modified bases. MD trajectories suggest that τm5s2U recognizes the codons initially by ‘wobble’ hydrogen bonding interactions, and then tRNALys might leave the explicit codon by a novel ‘single’ hydrogen bonding interaction in order to run the protein biosynthesis process smoothly. We propose this model as the ‘Foot-Step Model’ for codon recognition, in which the single hydrogen bond plays a crucial role. MD simulation results suggest that, tRNALys with τm5s2U and t6A recognizes AAA codon more preferably than AAG. Thus, these results reveal the consequences of τm5s2U and t6A in recognition of AAA/AAG codons in mitochondrial disease, MERRF.

Hydroxylation of a conserved tRNA modification establishes non-universal genetic code in echinoderm mitochondria.

The genetic code is not frozen but still evolving, which can result in the acquisition of 'dialectal' codons that deviate from the universal genetic code. RNA modifications in the anticodon region of tRNAs play a critical role in establishing such non-universal genetic codes. In echinoderm mitochondria, the AAA codon specifies asparagine instead of lysine. By analyzing mitochondrial (mt-) tRNALys isolated from the sea urchin (Mesocentrotus nudus), we discovered a novel modified nucleoside, hydroxy-N6-threonylcarbamoyladenosine (ht6A), 3' adjacent to the anticodon (position 37). Biochemical analysis revealed that ht6A37 has the ability to prevent mt-tRNALys from misreading AAA as lysine, thereby indicating that hydroxylation of N6-threonylcarbamoyladenosine (t6A) contributes to the establishment of the non-universal genetic code in echinoderm mitochondria.

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

Thio-Modification of tRNA at the Wobble Position as Regulator of the Kinetics of Decoding and Translocation on the Ribosome.

Uridine 34 (U34) at the wobble position of the tRNA anticodon is post-transcriptionally modified, usually to mcm5s2, mcm5, or mnm5. The lack of the mcm5 or s2 modification at U34 of tRNALys, tRNAGlu, and tRNAGln causes ribosome pausing at the respective codons in yeast. The pauses occur during the elongation step, but the mechanism that triggers ribosome pausing is not known. Here, we show how the s2 modification in yeast tRNALys affects mRNA decoding and tRNA-mRNA translocation. Using real-time kinetic analysis we show that mcm5-modified tRNALys lacking the s2 group has a lower affinity of binding to the cognate codon and is more efficiently rejected than the fully modified tRNALys. The lack of the s2 modification also slows down the rearrangements in the ribosome-EF-Tu-GDP-Pi-Lys-tRNALys complex following GTP hydrolysis by EF-Tu. Finally, tRNA-mRNA translocation is slower with the s2-deficient tRNALys. These observations explain the observed ribosome pausing at AAA codons during translation and demonstrate how the s2 modification helps to ensure the optimal translation rates that maintain proteome homeostasis of the cell.

Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code.

Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNALysUUU with hypermodified 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine–pyrimidine mismatches. We show that mnm5s2U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism.

In silico analysis of histone H3 gene expression during human brain development.

Precise regulation of chromatin structure is essential for proper development of higher eukaryotes, and methylation of histone H3 at lysine-27 (H3K27) by the Polycomb Repressive Complex 2 (PRC2) component EZH2 has emerged as an important and conserved mechanism to ensure silencing of developmentally regulated genes. Recurrent mutations within the histone H3 genes H3F3A and HIST1H3B that convert K27 to methionine (H3K27M) and disrupt the global H3K27 methylation landscape and PRC2-dependent silencing, have recently been identified in pediatric high-grade gliomas including Diffuse Intrinsic Pontine Glioma (DIPG) and Glioblastoma multiforme (GBM; Type IV glioma). These findings have generated renewed interest in the dynamics of histone genes and their expression, which have been difficult to study due to redundancy and high sequence homology within the H3 gene family. In this in silico study, we re-evaluated genomic organization of the human H3 gene family and expression of these genes in the human brain, utilizing public RNA-based sequence datasets for the human genome and brain development. We identified transcriptional activity from at least 17 protein-encoding H3 genes in the developing brain, comprising at least 14 canonical (H3.1)-like and 3 'replication-independent' (H3.3)-like forms, and encoding six distinct H3 isoforms. Transcripts for H3.3 genes including H3F3A show gradual decrease in abundance associated with developmental progression, whereas H3.1 transcripts including HIST1H3B tend to be strongly downregulated at an early prenatal stage and remain essentially silent thereafter. Twelve genes, including members of both H3.1 and H3.3 classes, contain a K27-AAG codon that is mutable to that for M (ATG), whereas the remaining contain the alternative, AAA codon for K at this position. H3F3A is the only H3.3-like gene containing the K27-AAG codon, whereas HIST1H3B is among ten H3.1-like genes containing this codon. This data indicates that, in the early developing human brain, HIST1H3B constitutes the largest proportion of H3.1 transcripts among H3.1 isoforms. We suggest that the apparent overrepresentation of K27M mutations in H3F3A relative to other H3 isoforms may result from its uniqueness among H3.3s for the K27-AAG codon and the functional relationship between H3.3 and PRC2, whereas overrepresentation of K27M mutations in HIST1H3B may be a product of strong relative expression of this gene in the early developing brain.

Elongator-dependent modification of cytoplasmic tRNALysUUU is required for mitochondrial function under stress conditions.

To gain a wider view of the pathways that regulate mitochondrial function, we combined the effect of heat stress on respiratory capacity with the discovery potential of a genome-wide screen in Saccharomyces cerevisiae. We identified 105 new genes whose deletion impairs respiratory growth at 37°C by interfering with processes such as transcriptional regulation, ubiquitination and cytosolic tRNA wobble uridine modification via 5-methoxycarbonylmethyl-2-thiouridine formation. The latter process, specifically required for efficient decoding of AA-ending codons under stress conditions, was covered by multiple genes belonging to the Elongator (e.g. ELP3) and urmylation (e.g., NCS6) pathways. ELP3 or NCS6 deletants had impaired mitochondrial protein synthesis. Their respiratory deficiency was selectively rescued by overexpression of tRNALysUUU as well by overexpression of genes (BCK1 and HFM1) with a strong bias for the AAA codon read by this tRNA. These data extend the mitochondrial regulome, demonstrate that heat stress can impair respiration by disturbing cytoplasmic translation of proteins critically involved in mitochondrial function and document, for the first time, the involvement in such process of the Elongator and urmylation pathways. Given the conservation of these pathways, the present findings may pave the way to a better understanding of the human mitochondrial regulome in health and disease.

Ribosomes slide on lysine-encoding homopolymeric A stretches.

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome 'sliding' represents an unexpected type of ribosome movement possible during translation.

Basic concepts and potential applications of genetics and genomics for cardiovascular and stroke clinicians: a scientific statement from the American Heart Association.

Although genetics and genomics play an increasingly large role in the practice of medicine, the clinical care of patients suffering from cardiovascular disease or stroke has not been significantly affected. This is despite the tremendous strides being made to understand the genetic basis of both rare and common cardiovascular and stroke disorders through techniques such as genome-wide association studies (GWASs) and next-generation sequencing studies. Much of this knowledge remains to be translated to the clinic and must be subjected to clinical trials to ensure patient safety and a meaningful impact on clinical outcomes. However, even if this knowledge were to be successfully implemented into clinical practice, a potential barrier to widespread adoption is a lack of familiarity with basic concepts of genetics and genomics. Another concern is the possibility of the emergence of a significant gap in clinical care provided by practitioners who are informed about the clinical use of genetics and genomics knowledge and those who are not. Thus, there is a critical need to foster genetics/genomics literacy among all involved in the care of cardiovascular and stroke patients because it can be expected that these topics will transform the way medicine is practiced. The purpose of this document is to serve as a resource for practitioners in cardiovascular and stroke medicine on the application of genetics and genomics to patient care. Although not exhaustive, it contains an overview of the field written specifically to be accessible and relevant to practitioners. It also refers to additional educational materials available in the literature, in textbooks, and on the Internet. (Because this article is intended to be primarily educational in nature, rather than providing a review of the literature, citations are limited to a small number of research articles and reviews of exceptional interest.) It recommends a core knowledge base with …