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Editorial

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4 pages, 178 KiB

Open AccessEditorial

Synthetic DNA and RNA Programming

by Patrick O’Donoghue and Ilka U. Heinemann

Genes 2019, 10(7), 523; https://doi.org/10.3390/genes10070523 - 11 Jul 2019

Cited by 1 | Viewed by 4573

Abstract

Synthetic biology is a broad and emerging discipline that capitalizes on recent advances in molecular biology, genetics, protein and RNA engineering as well as omics technologies. Together these technologies have transformed our ability to reveal the biology of the cell and the molecular [...] Read more.

Synthetic biology is a broad and emerging discipline that capitalizes on recent advances in molecular biology, genetics, protein and RNA engineering as well as omics technologies. Together these technologies have transformed our ability to reveal the biology of the cell and the molecular basis of disease. This Special Issue on “Synthetic RNA and DNA Programming” features original research articles and reviews, highlighting novel aspects of basic molecular biology and the molecular mechanisms of disease that were uncovered by the application and development of novel synthetic biology-driven approaches. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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Research

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12 pages, 2680 KiB

Open AccessArticle

Bacterial Aspartyl-tRNA Synthetase Has Glutamyl-tRNA Synthetase Activity

by Udumbara M. Rathnayake and Tamara L. Hendrickson

Genes 2019, 10(4), 262; https://doi.org/10.3390/genes10040262 - 1 Apr 2019

Cited by 4 | Viewed by 4080

Abstract

The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, [...] Read more.

The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNA^Asn via Asp-tRNA^Asn. This intermediate is produced by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) that has retained its ability to also generate Asp-tRNA^Asp. Here we report the discovery that ND-AspRS and its discriminating counterpart, AspRS, are also capable of specifically producing Glu-tRNA^Glu, without producing misacylated tRNAs like Glu-tRNA^Asn, Glu-tRNA^Asp, or Asp-tRNA^Glu, thus maintaining the fidelity of the genetic code. Consequently, bacterial AspRSs have glutamyl-tRNA synthetase-like activity that does not contaminate the proteome via amino acid misincorporation. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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11 pages, 4189 KiB

Open AccessArticle

Development of a Transformation Method for Metschnikowia borealis and other CUG-Serine Yeasts

by Zachary B. Gordon, Maximillian P.M. Soltysiak, Christopher Leichthammer, Jasmine A. Therrien, Rebecca S. Meaney, Carolyn Lauzon, Matthew Adams, Dong Kyung Lee, Preetam Janakirama, Marc-André Lachance and Bogumil J. Karas

Genes 2019, 10(2), 78; https://doi.org/10.3390/genes10020078 - 23 Jan 2019

Cited by 7 | Viewed by 5154

Abstract

Yeasts belonging to the Metschnikowia genus are particularly interesting for the unusual formation of only two needle-shaped ascospores during their mating cycle. Presently, the meiotic process that can lead to only two spores from a diploid zygote is poorly understood. The expression of [...] Read more.

Yeasts belonging to the Metschnikowia genus are particularly interesting for the unusual formation of only two needle-shaped ascospores during their mating cycle. Presently, the meiotic process that can lead to only two spores from a diploid zygote is poorly understood. The expression of fluorescent nuclear proteins should allow the meiotic process to be visualized in vivo; however, no large-spored species of Metschnikowia has ever been transformed. Accordingly, we aimed to develop a transformation method for Metschnikowia borealis, a particularly large-spored species of Metschnikowia, with the goal of enabling the genetic manipulations required to study biological processes in detail. Genetic analyses confirmed that M. borealis, and many other Metschnikowia species, are CUG-Ser yeasts. Codon-optimized selectable markers lacking CUG codons were used to successfully transform M. borealis by electroporation and lithium acetate, and transformants appeared to be the result of random integration. Mating experiments confirmed that transformed-strains were capable of generating large asci and undergoing recombination. Finally, random integration was used to transform an additional 21 yeast strains, and all attempts successfully generated transformants. The results provide a simple method to transform many yeasts from an array of different clades and can be used to study or develop many species for various applications. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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14 pages, 2402 KiB

Open AccessArticle

Acceptor Stem Differences Contribute to Species-Specific Use of Yeast and Human tRNA^Ser

by Matthew D. Berg, Julie Genereaux, Yanrui Zhu, Safee Mian, Gregory B. Gloor and Christopher J. Brandl

Genes 2018, 9(12), 612; https://doi.org/10.3390/genes9120612 - 7 Dec 2018

Cited by 9 | Viewed by 4704

Abstract

The molecular mechanisms of translation are highly conserved in all organisms indicative of a single evolutionary origin. This includes the molecular interactions of tRNAs with their cognate aminoacyl-tRNA synthetase, which must be precise to ensure the specificity of the process. For many tRNAs, [...] Read more.

The molecular mechanisms of translation are highly conserved in all organisms indicative of a single evolutionary origin. This includes the molecular interactions of tRNAs with their cognate aminoacyl-tRNA synthetase, which must be precise to ensure the specificity of the process. For many tRNAs, the anticodon is a major component of the specificity. This is not the case for the aminoacylation of alanine and serine to their cognate tRNAs. Rather, aminoacylation relies on other features of the tRNA. For tRNA^Ser, a key specificity feature is the variable arm, which is positioned between the anticodon arm and the T-arm. The variable arm is conserved from yeast to human. This work was initiated to determine if the structure/function of tRNA^Ser has been conserved from Saccharomyces cerevisiae to human. We did this by detecting mistranslation in yeast cells with tRNA^Ser derivatives having the UGA anticodon converted to UGG for proline. Despite being nearly identical in everything except the acceptor stem, human tRNA^Ser is less active than yeast tRNA^Ser. A chimeric tRNA with the human acceptor stem and other sequences from the yeast molecule acts similarly to the human tRNA^Ser. The 3:70 base pair in the acceptor stem (C:G in yeast and A:U in humans) is a prime determinant of the specificity. Consistent with the functional difference of yeast and human tRNA^Ser resulting from subtle changes in the specificity of their respective SerRS enzymes, the functionality of the human and chimeric tRNA^Ser_UGG molecules was enhanced when human SerRS was introduced into yeast. Residues in motif 2 of the aminoacylation domain of SerRS likely participated in the species-specific differences. Trp290 in yeast SerRS (Arg313 in humans) found in motif 2 is proximal to base 70 in models of the tRNA-synthetase interaction. Altering this motif 2 sequence of hSerRS to the yeast sequence decreases the activity of the human enzyme with human tRNA^Ser, supporting the coadaptation of motif 2 loop–acceptor stem interactions. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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17 pages, 1228 KiB

Open AccessArticle

Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair

by David G. Schwark, Margaret A. Schmitt and John D. Fisk

Genes 2018, 9(11), 546; https://doi.org/10.3390/genes9110546 - 12 Nov 2018

Cited by 21 | Viewed by 4001

Abstract

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting [...] Read more.

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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16 pages, 2831 KiB

Open AccessArticle

Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

by Takuya Umehara, Saori Kosono, Dieter Söll and Koji Tamura

Genes 2018, 9(10), 473; https://doi.org/10.3390/genes9100473 - 28 Sep 2018

Cited by 11 | Viewed by 4606

Abstract

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia [...] Read more.

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which N^ε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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16 pages, 3207 KiB

Open AccessFeature PaperArticle

Phosphorylation-Dependent Inhibition of Akt1

by Nileeka Balasuriya, McShane McKenna, Xuguang Liu, Shawn S. C. Li and Patrick O’Donoghue

Genes 2018, 9(9), 450; https://doi.org/10.3390/genes9090450 - 7 Sep 2018

Cited by 37 | Viewed by 8723

Abstract

Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology [...] Read more.

Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1^T308 and showed four-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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12 pages, 3243 KiB

Open AccessArticle

Genome-Wide Quantification of the Effect of Gene Overexpression on Escherichia coli Growth

by Hao Chen, Sumana Venkat, Jessica Wilson, Paige McGuire, Abigail L. Chang, Qinglei Gan and Chenguang Fan

Genes 2018, 9(8), 414; https://doi.org/10.3390/genes9080414 - 16 Aug 2018

Cited by 11 | Viewed by 4392

Abstract

Recombinant protein production plays an essential role in both biological studies and pharmaceutical production. Escherichia coli is one of the most favorable hosts for this purpose. Although a number of strategies for optimizing protein production have been developed, the effect of gene overexpression [...] Read more.

Recombinant protein production plays an essential role in both biological studies and pharmaceutical production. Escherichia coli is one of the most favorable hosts for this purpose. Although a number of strategies for optimizing protein production have been developed, the effect of gene overexpression on host cell growth has been much less studied. Here, we performed high-throughput tests on the E. coli a complete set of E. coli K-12 ORF archive (ASKA) collection to quantify the effects of overexpressing individual E. coli genes on its growth. The results indicated that overexpressing membrane-associated proteins or proteins with high abundances of branched-chain amino acids tended to impair cell growth, the latter of which could be remedied by amino acid supplementation. Through this study, we expect to provide an index for a fast pre-study estimate of host cell growth in order to choose proper rescuing approaches when working with different proteins. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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12 pages, 1967 KiB

Open AccessArticle

MiRAR—miRNA Activity Reporter for Living Cells

by Matthew A. Turk, Christina Z. Chung, Emad Manni, Stephanie A. Zukowski, Anish Engineer, Yasaman Badakhshi, Yumin Bi and Ilka U. Heinemann

Genes 2018, 9(6), 305; https://doi.org/10.3390/genes9060305 - 19 Jun 2018

Cited by 11 | Viewed by 5476

Abstract

microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time [...] Read more.

microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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Review

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18 pages, 2081 KiB

Open AccessReview

The Role of 3′ to 5′ Reverse RNA Polymerization in tRNA Fidelity and Repair

by Allan W. Chen, Malithi I. Jayasinghe, Christina Z. Chung, Bhalchandra S. Rao, Rosan Kenana, Ilka U. Heinemann and Jane E. Jackman

Genes 2019, 10(3), 250; https://doi.org/10.3390/genes10030250 - 26 Mar 2019

Cited by 13 | Viewed by 5703

Abstract

The tRNA^His guanylyltransferase (Thg1) superfamily includes enzymes that are found in all three domains of life that all share the common ability to catalyze the 3′ to 5′ synthesis of nucleic acids. This catalytic activity, which is the reverse of all other [...] Read more.

The tRNA^His guanylyltransferase (Thg1) superfamily includes enzymes that are found in all three domains of life that all share the common ability to catalyze the 3′ to 5′ synthesis of nucleic acids. This catalytic activity, which is the reverse of all other known DNA and RNA polymerases, makes this enzyme family a subject of biological and mechanistic interest. Previous biochemical, structural, and genetic investigations of multiple members of this family have revealed that Thg1 enzymes use the 3′ to 5′ chemistry for multiple reactions in biology. Here, we describe the current state of knowledge regarding the catalytic features and biological functions that have been so far associated with Thg1 and its homologs. Progress toward the exciting possibility of utilizing this unusual protein activity for applications in biotechnology is also discussed. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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16 pages, 1703 KiB

Open AccessReview

Alternative Biochemistries for Alien Life: Basic Concepts and Requirements for the Design of a Robust Biocontainment System in Genetic Isolation

by Christian Diwo and Nediljko Budisa

Genes 2019, 10(1), 17; https://doi.org/10.3390/genes10010017 - 28 Dec 2018

Cited by 22 | Viewed by 6633

Abstract

The universal genetic code, which is the foundation of cellular organization for almost all organisms, has fostered the exchange of genetic information from very different paths of evolution. The result of this communication network of potentially beneficial traits can be observed as modern [...] Read more.

The universal genetic code, which is the foundation of cellular organization for almost all organisms, has fostered the exchange of genetic information from very different paths of evolution. The result of this communication network of potentially beneficial traits can be observed as modern biodiversity. Today, the genetic modification techniques of synthetic biology allow for the design of specialized organisms and their employment as tools, creating an artificial biodiversity based on the same universal genetic code. As there is no natural barrier towards the proliferation of genetic information which confers an advantage for a certain species, the naturally evolved genetic pool could be irreversibly altered if modified genetic information is exchanged. We argue that an alien genetic code which is incompatible with nature is likely to assure the inhibition of all mechanisms of genetic information transfer in an open environment. The two conceivable routes to synthetic life are either de novo cellular design or the successive alienation of a complex biological organism through laboratory evolution. Here, we present the strategies that have been utilized to fundamentally alter the genetic code in its decoding rules or its molecular representation and anticipate future avenues in the pursuit of robust biocontainment. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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15 pages, 1662 KiB

Open AccessReview

Cyclic Peptides: Promising Scaffolds for Biopharmaceuticals

by Donghyeok Gang, Do Wook Kim and Hee-Sung Park

Genes 2018, 9(11), 557; https://doi.org/10.3390/genes9110557 - 16 Nov 2018

Cited by 80 | Viewed by 9488

Abstract

To date, small molecules and macromolecules, including antibodies, have been the most pursued substances in drug screening and development efforts. Despite numerous favorable features as a drug, these molecules still have limitations and are not complementary in many regards. Recently, peptide-based chemical structures [...] Read more.

To date, small molecules and macromolecules, including antibodies, have been the most pursued substances in drug screening and development efforts. Despite numerous favorable features as a drug, these molecules still have limitations and are not complementary in many regards. Recently, peptide-based chemical structures that lie between these two categories in terms of both structural and functional properties have gained increasing attention as potential alternatives. In particular, peptides in a circular form provide a promising scaffold for the development of a novel drug class owing to their adjustable and expandable ability to bind a wide range of target molecules. In this review, we discuss recent progress in methodologies for peptide cyclization and screening and use of bioactive cyclic peptides in various applications. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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15 pages, 1012 KiB

Open AccessReview

Versatility of Synthetic tRNAs in Genetic Code Expansion

by Kyle S. Hoffman, Ana Crnković and Dieter Söll

Genes 2018, 9(11), 537; https://doi.org/10.3390/genes9110537 - 7 Nov 2018

Cited by 13 | Viewed by 6648

Abstract

Transfer RNA (tRNA) is a dynamic molecule used by all forms of life as a key component of the translation apparatus. Each tRNA is highly processed, structured, and modified, to accurately deliver amino acids to the ribosome for protein synthesis. The tRNA molecule [...] Read more.

Transfer RNA (tRNA) is a dynamic molecule used by all forms of life as a key component of the translation apparatus. Each tRNA is highly processed, structured, and modified, to accurately deliver amino acids to the ribosome for protein synthesis. The tRNA molecule is a critical component in synthetic biology methods for the synthesis of proteins designed to contain non-canonical amino acids (ncAAs). The multiple interactions and maturation requirements of a tRNA pose engineering challenges, but also offer tunable features. Major advances in the field of genetic code expansion have repeatedly demonstrated the central importance of suppressor tRNAs for efficient incorporation of ncAAs. Here we review the current status of two fundamentally different translation systems (TSs), selenocysteine (Sec)- and pyrrolysine (Pyl)-TSs. Idiosyncratic requirements of each of these TSs mandate how their tRNAs are adapted and dictate the techniques used to select or identify the best synthetic variants. Full article

(This article belongs to the Special Issue Synthetic DNA and RNA Programming)

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