Replacement of All Arginine Residues with Canavanine in MazF-bs mRNA Interferase Changes Its Specificity

Ishida, Yojiro; Park, Jung‐Ho; Mao, Lili; Yamaguchi, Yoshihiro; Inouye, Masayori

doi:10.1074/jbc.m112.434969

Cited by 16 publications

(12 citation statements)

References 24 publications

(17 reference statements)

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“…It is possible that a pyrimidine base could be accommodated inside a purine binding pocket without any steric hindrance, thereby resulting in the cleavage of RNA at such locations, albeit with a lower efficiency. Recently, it has been shown that the replacement of all Arg residues with canavanine (a toxic Arg analog) in B. subtilis MazF changes its substrate specificity to a six-base target sequence U^ACAUA (Ishida et al, 2013). Examination of the location of all Arg residues present in the structure of the MazF-RNA complex suggests that the change in substrate specificity is likely due to overall conformational changes occurring within the MazF dimer, mainly due to the substitution of Arg81 and Arg87 present at the dimer interface, Arg25 at the cleavage site and Arg37 present near the RNA binding pocket involving recognition of the sixth nucleotide.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis of mRNA Recognition and Cleavage by Toxin MazF and Its Regulation by Antitoxin MazE in Bacillus subtilis

et al. 2013

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SUMMARY MazF is an mRNA interferase, which, upon activation during stress conditions, cleaves mRNAs in a sequence-specific manner, resulting in cellular growth arrest. During normal growth conditions, the MazF toxin is inactivated through binding to its cognate antitoxin, MazE. How MazF specifically recognizes its mRNA target and carries out cleavage and how the formation of the MazE-MazF complex inactivates MazF remain unclear. We present crystal structures of MazF in complex with mRNA substrate and antitoxin MazE in Bacillus subtilis. The structure of MazF in complex with uncleavable UUdUACAUAA RNA substrate defines the molecular basis underlying the sequence-specific recognition of UACAU and the role of residues involved in the cleavage through site-specific mutational studies. The structure of the heterohexameric (MazF)2-(MazE)2-(MazF)2 complex in Bacillus subtilis, supplemented by mutational data, demonstrates that the positioning of the C-terminal helical segment of MazE within the RNA-binding channel of the MazF dimer prevents mRNA binding and cleavage by MazF.

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Section: Discussionmentioning

confidence: 99%

Structural Basis of mRNA Recognition and Cleavage by Toxin MazF and Its Regulation by Antitoxin MazE in Bacillus subtilis

et al. 2013

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“…This simplifies the residue-specific incorporation of Arg analogs compared to other expression systems that require further depletion strategies 29,30 . The presented cell-free approach circumvents the inherent limitations of in vivo approaches that are due to Can toxicity, or the strong dependency on mRNA sequence in single protein production strategies 24,31 . Contrary to the employed in vitro system, in vivo cleavage of Can to homoserine and hydroxyguanidine does occur 31 .…”

Section: Representative Resultsmentioning

confidence: 99%

“…Due to the resulting toxicity, expression of canavanine containing proteins in Escherichia coli (E. coli) and other common expression hosts remains a challenge. For these reasons, complete in vivo incorporation of Can at all Arg positions has appropriately been confirmed only once 24 , using an elaborated single-protein production system. However, Can has been proposed as an anti-cancer agent [25][26][27] , and as a stimulator for autoimmune diseases in humans 28 .…”

Section: Introductionmentioning

confidence: 99%

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an <em>Escherichia coli</em> Cell-free Transcription-translation System

Worst

Exner

Simone

et al. 2016

JoVE

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The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

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“…Our method enables the efficient production of target canavanine proteins while avoiding the limitations of in vivo systems, such as canavanine toxicity, or the strong dependency on mRNA sequence in strategies based on the single protein production (SPP) method. 16 The general applicability and usability make our method a potent tool for further research in canavanine toxicity and therapeutic application.…”

mentioning

confidence: 99%

“…15 Not surprisingly, only one instance of a protein with confirmed full replacement of arginines by canavanine has been reported so far, using an elaborate single-protein production system. 16 The problem of high toxicity can be circumvented by the use of sophisticated in vitro expression systems. Such cell-free systems play an increasing role in a number of biotechnology and proteomic applications.…”

mentioning

confidence: 99%

Cell-free expression with the toxic amino acid canavanine

Worst

Exner

Simone

et al. 2015

Bioorganic & Medicinal Chemistry Letters

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Replacement of All Arginine Residues with Canavanine in MazF-bs mRNA Interferase Changes Its Specificity

Cited by 16 publications

References 24 publications

Structural Basis of mRNA Recognition and Cleavage by Toxin MazF and Its Regulation by Antitoxin MazE in Bacillus subtilis

Structural Basis of mRNA Recognition and Cleavage by Toxin MazF and Its Regulation by Antitoxin MazE in Bacillus subtilis

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an <em>Escherichia coli</em> Cell-free Transcription-translation System

Cell-free expression with the toxic amino acid canavanine

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