scholarly journals Nonsense suppression by near-cognate tRNAs employs alternative base pairing at codon positions 1 and 3

2015 ◽  
Vol 112 (10) ◽  
pp. 3038-3043 ◽  
Author(s):  
Bijoyita Roy ◽  
John D. Leszyk ◽  
David A. Mangus ◽  
Allan Jacobson
Keyword(s):  
Amino Acids ◽  
Full Length ◽  
Nonsense Suppression ◽  
Truncated Protein ◽  
Premature Termination Codons ◽  
Codon Positions ◽  
Nonsense Codons ◽  
Readthrough Protein ◽  
A Site ◽  
Cognate Trna

Premature termination codons (PTCs) in an mRNA ORF inactivate gene function by causing production of a truncated protein and destabilization of the mRNA. Readthrough of a PTC allows ribosomal A-site insertion of a near-cognate tRNA, leading to synthesis of a full-length protein from otherwise defective mRNA. To understand the mechanism of such nonsense suppression, we developed a yeast system that allows purification and sequence analysis of full-length readthrough products arising as a consequence of endogenous readthrough or the compromised termination fidelity attributable to the loss of Upf (up-frameshift) factors, defective release factors, or the presence of the aminoglycoside gentamicin. Unlike classical “wobble” models, our analyses showed that three of four possible near-cognate tRNAs could mispair at position 1 or 3 of nonsense codons and that, irrespective of whether readthrough is endogenous or induced, the same sets of amino acids are inserted. We identified the insertion of Gln, Tyr, and Lys at UAA and UAG, whereas Trp, Arg, and Cys were inserted at UGA, and the frequency of insertion of individual amino acids was distinct for specific nonsense codons and readthrough-inducing agents. Our analysis suggests that the use of genetic or chemical means to increase readthrough does not promote novel or alternative mispairing events; rather, readthrough effectors cause quantitative enhancement of endogenous mistranslation events. Knowledge of the amino acids incorporated during readthrough not only elucidates the decoding process but also may allow predictions of the functionality of readthrough protein products.

scholarly journals Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

2016 ◽  
Vol 113 (44) ◽  
pp. 12508-12513 ◽  
Author(s):  
Bijoyita Roy ◽  
Westley J. Friesen ◽  
Yuki Tomizawa ◽  
John D. Leszyk ◽  
Jin Zhuo ◽  
...  
Keyword(s):  
Premature Termination Codon ◽  
Nonsense Suppression ◽  
Base Pairs ◽  
Inherited Disorders ◽  
Nonsense Mutations ◽  
Nonsense Codon ◽  
Codon Positions ◽  
A Site ◽  
Cognate Trna ◽  
Selection Of

A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren’s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren’s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

scholarly journals Molecular Insights into Determinants of Translational Readthrough and Implications for Nonsense Suppression Approaches

2020 ◽  
Vol 21 (24) ◽  
pp. 9449
Author(s):  
Silvia Lombardi ◽  
Maria Francesca Testa ◽  
Mirko Pinotti ◽  
Alessio Branchini
Keyword(s):  
Therapeutic Strategy ◽  
Low Frequency ◽  
External Factors ◽  
Full Length ◽  
Nonsense Suppression ◽  
Coagulation Disorders ◽  
Nonsense Mutations ◽  
Premature Termination Codons ◽  
Translational Readthrough ◽  
Molecular Determinants

The fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense and stop codons. However, premature termination codons (PTCs) arising from mutations may, at low frequency, be misrecognized and result in PTC suppression, named ribosome readthrough, with production of full-length proteins through the insertion of a subset of amino acids. Since some drugs have been identified as readthrough inducers, this fidelity drawback has been explored as a therapeutic approach in several models of human diseases caused by nonsense mutations. Here, we focus on the mechanisms driving translation in normal and aberrant conditions, the potential fates of mRNA in the presence of a PTC, as well as on the results obtained in the research of efficient readthrough-inducing compounds. In particular, we describe the molecular determinants shaping the outcome of readthrough, namely the nucleotide and protein context, with the latter being pivotal to produce functional full-length proteins. Through the interpretation of experimental and mechanistic findings, mainly obtained in lysosomal and coagulation disorders, we also propose a scenario of potential readthrough-favorable features to achieve relevant rescue profiles, representing the main issue for the potential translatability of readthrough as a therapeutic strategy.

scholarly journals Recent Advances in Chiral Analysis of Proteins and Peptides

Separations ◽  
2021 ◽  
Vol 8 (8) ◽  
pp. 112
Author(s):  
Marine Morvan ◽  
Ivan Mikšík
Keyword(s):  
Amino Acids ◽  
Stationary Phases ◽  
Enzymatic Digestion ◽  
Chiral Separations ◽  
Biological Compounds ◽  
Health And Disease ◽  
Electrophoretic Techniques ◽  
A Site ◽  
Future Work ◽  
Proteins And Peptides

Like many biological compounds, proteins are found primarily in their homochiral form. However, homochirality is not guaranteed throughout life. Determining their chiral proteinogenic sequence is a complex analytical challenge. This is because certain D-amino acids contained in proteins play a role in human health and disease. This is the case, for example, with D-Asp in elastin, β-amyloid and α-crystallin which, respectively, have an action on arteriosclerosis, Alzheimer's disease and cataracts. Sequence-dependent and sequence-independent are the two strategies for detecting the presence and position of D-amino acids in proteins. These methods rely on enzymatic digestion by a site-specific enzyme and acid hydrolysis in a deuterium or tritium environment to limit the natural racemization of amino acids. In this review, chromatographic and electrophoretic techniques, such as LC, SFC, GC and CE, will be recently developed (2018–2020) for the enantioseparation of amino acids and peptides. For future work, the discovery and development of new chiral stationary phases and derivatization reagents could increase the resolution of chiral separations.

scholarly journals In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catalyzed by the Salmonella Hin Recombinase

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1649-1663
Author(s):  
Oliver Z Nanassy ◽  
Kelly T Hughes
Keyword(s):  
Amino Acids ◽  
Biochemical Analysis ◽  
Mutational Analysis ◽  
Recombination Reaction ◽  
Recombination Site ◽  
Dna Inversion ◽  
Hin Recombinase ◽  
One Step ◽  
A Site

Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

scholarly journals Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

2021 ◽  
Vol 16 (4) ◽  
pp. 766-774
Author(s):  
Jeffery M. Tharp ◽  
Oscar Vargas-Rodriguez ◽  
Alanna Schepartz ◽  
Dieter Söll
Keyword(s):  
Amino Acids ◽  
Noncanonical Amino Acids ◽  
Genetic Encoding ◽  
Nonsense Codons

NonO, a non-POU-domain-containing, octamer-binding protein, is the mammalian homolog of Drosophila nonAdiss

1993 ◽  
Vol 13 (9) ◽  
pp. 5593-5603
Author(s):  
Y S Yang ◽  
J H Hanke ◽  
L Carayannopoulos ◽  
C M Craft ◽  
J D Capra ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Protein Binding ◽  
Aromatic Amino Acids ◽  
Courtship Song ◽  
Dna Binding Domain ◽  
Binding Motifs ◽  
Dna And Rna ◽  
Pou Domain ◽  
A Site

We have cloned the ubiquitous form of an octamer-binding, 60-kDa protein (NonO) that appears to be the mammalian equivalent of the Drosophila visual and courtship song behavior protein, no-on-transient A/dissonance (nonAdiss). A region unprecedently rich in aromatic amino acids containing two ribonuclear protein binding motifs is highly conserved between the two proteins. A ubiquitous form of NonO is present in all adult tissues, whereas lymphocytes and retina express unique forms of NonO mRNA. The ubiquitous form contains a potential helix-turn-helix motif followed by a highly charged region but differs from prototypic octamer-binding factors by lacking the POU DNA-binding domain. In addition to its conventional octamer duplex-binding, NonO binds single-stranded DNA and RNA at a site independent of the duplex site.

Elongation factor EF-1 alpha gene dosage alters translational fidelity in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (10) ◽  
pp. 4571-4575
Author(s):  
J M Song ◽  
S Picologlou ◽  
C M Grant ◽  
M Firoozan ◽  
M F Tuite ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Dosage ◽  
Elongation Factor ◽  
Nonsense Suppression ◽  
Translational Fidelity ◽  
Genes Encoding ◽  
Alpha Gene ◽  
Nonsense Codons

Changes in the dosage of genes encoding elongation factor EF-1 alpha were shown to cause parallel changes in the misreading of nonsense codons. Higher amounts of EF-1 alpha were correlated with increased nonsense suppression, suggesting that the level of EF-1 alpha is critically involved in translational fidelity.

scholarly journals Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Matthew T Parker ◽  
Katarzyna Knop ◽  
Anna V Sherwood ◽  
Nicholas J Schurch ◽  
Katarzyna Mackinnon ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Single Molecule ◽  
Tail Length ◽  
Transcript Abundance ◽  
Mrna Processing ◽  
Full Length ◽  
Transcription Start Sites ◽  
Model Plant Arabidopsis Thaliana ◽  
Defective Rna ◽  
A Site

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 3′ end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

Association of the IVR Gene with Virus Localization and Resistance

1995 ◽  
Author(s):  
Gad Loebenstein ◽  
William Dawson ◽  
Abed Gera
Keyword(s):  
Amino Acids ◽  
Coat Protein ◽  
Plant Viruses ◽  
Full Length ◽  
N Gene ◽  
Complete Suppression ◽  
Coat Proteins ◽  
Nicotiana Glutinosa ◽  
Nicotiana Debneyi ◽  
Full Length Gene

We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi. During the present grant we found that TMV production is enhanced in protoplasts and plants of local lesion responding tobacco cultivars exposed to 35oC, parallel to an almost complete suppression of the production of IVR. We also found that IVR is associated with resistance mechanisms in pepper cultivars. We succeeded to clone the IVR gene. In the first attempt we isolated a clone - "101" which had a specific insert of 372 bp (the full length gene for the IVR protein of 23 kD should be around 700 bp). However, attempts to isolate the full length gene did not give clear cut results, and we decided not to continue with this clone. The amino acid sequence of the N-terminus of IVR was determined and an antiserum was prepared against a synthetic peptide representing amino acids residues 1-20 of IVR. Using this antiserum as well as our polyclonal antiserum to IVR a new clone NC-330 was isolated using lamba-ZAP library. This NC-330 clone has an insert of about 1 kB with an open reading frame of 596 bp. This clone had 86.6% homology with the first 15 amino acids of the N-terminal part of IVR and 61.6% homology with the first 23 amino acids of IVR. In the QIA expression system and western blotting of the expressed protein, a clear band of about 21 kD was obtained with IVR antiserum. This clone was used for transformation of Samsun tobacco plants and we have presently plantlets which were rooted on medium containing kanamycin. Hybridization with this clone was also obtained with RNA from induced resistant tissue of Samsun NN but not with RNA from healthy control tissue of Samsun NN, or infected or healthy tissue of Samsun. This further strengthens the previous data that the NC 330 clone codes for IVR. In the U.S. it was shown that IVR is induced in plants containing the N' gene when infected with mutants of TMV that elicit the HR. This is a defined system in which the elicitor is known to be due to permutations of the coat protein which can vary in elicitor strength. The objective was to understand how IVR synthesis is induced after recognition of elicitor coat protein in the signal transduction pathway that leads to HR. We developed systems to manipulate induction of IVR by modifying the elicitor and are using these elicitor molecules to isolate the corresponding plant receptor molecules. A "far-western" procedure was developed that found a protein from N' plants that specifically bind to elicitor coat proteins. This protein is being purified and sequenced. This objective has not been completed and is still in progress. We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi.

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