Purification and Use of tRNA for Enzymatic Post-translational Addition of Amino Acids to Proteins

Avcilar-Kucukgoze, Irem; Gamper, Howard; Hou, Ya‐Ming; Kashina, Anna

doi:10.1016/j.xpro.2020.100207

Cited by 14 publications

(7 citation statements)

References 17 publications

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“…To further validate that 10-F05 inhibits MiaA in cells, we proceeded to purify the modified tRNA from E. coli and enzymatically digested it into individual ribonucleosides using established methods. 38,39 Subsequently, we quantified the presence of i 6 A and ms 2 i 6 A (from i 6 A by the action of MiaB) using LC-MS analysis ( Figure 7C, top ). The i 6 A modification level was extremely low and below the detectable limit of our LC-MS instrument.…”

Section: Resultsmentioning

confidence: 99%

Phenotypic screening of covalent compound libraries identifies chloromethyl ketone antibiotics and MiaA as a new target

Jin,

Jana,

Abbasov

et al. 2024

Preprint

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The emerging antibiotic resistance requires the development of new antibiotics working on novel bacterial targets. Here, we reported an antibiotic discovery workflow by combining the cysteine-reactive compound library phenotypic screening with activity-based protein profiling, which enables the rapid identification of lead compounds as well as new druggable targets in pathogens. Compounds featuring chloromethyl ketone scaffolds exhibited a notably high hit rate against both gram-negative and gram-positive bacterial strains, but not the more commonly used warheads such as acrylamide or chloroacetamide. Target identification of the lead compound, 10-F05, revealed that its primary targets in S. flexneri are FabH Cys112 and MiaA Cys273. We validated the target relevance through biochemical and genetic interactions. Mechanistic studies revealed modification of MiaA by 10-F05 impair substrate tRNA binding, leading to decreased bacterial stress resistance and virulence. Our findings underscore chloromethyl ketone as a novel antibacterial warhead in covalent antibiotic design. The study showcases that combining covalent compound library phenotypic screening with chemoproteomics is an efficient way to identify new drug targets as well as lead compounds, with the potential to open new research directions in drug discovery and chemical biology.

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Section: Resultsmentioning

confidence: 99%

Phenotypic screening of covalent compound libraries identifies chloromethyl ketone antibiotics and MiaA as a new target

Jin,

Jana,

Abbasov

et al. 2024

Preprint

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“…Bulk tRNA from Mycoplasma capricolum subsp. capri was isolated according to the method described by Avcilar-Kucukgoze et al with minor alterations. Mycoplasma capricolum subsp.…”

Section: Methodsmentioning

confidence: 99%

Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium

et al. 2023

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Cell-free expression (CFE) systems are fundamental to reconstituting metabolic pathways in vitro toward the construction of a synthetic cell. Although an Escherichia coli-based CFE system is well-established, simpler model organisms are necessary to understand the principles behind life-like behavior. Here, we report the successful creation of a CFE system derived from JCVI-syn3A (Syn3A), the minimal synthetic bacterium. Previously, high ribonuclease activity in Syn3A lysates impeded the establishment of functional CFE systems. Now, we describe how an unusual cell lysis method (nitrogen decompression) yielded Syn3A lysates with reduced ribonuclease activity that supported in vitro expression. To improve the protein yields in the Syn3A CFE system, we optimized the Syn3A CFE reaction mixture using an active machine learning tool. The optimized reaction mixture improved the CFE 3.2-fold compared to the preoptimized condition. This is the first report of a functional CFE system derived from a minimal synthetic bacterium, enabling further advances in bottom-up synthetic biology.

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“…The aqueous phase was collected, and RNA precipitated in 2.5 volumes of ethanol and 0.1 volume of 2.5 M NaOAc pH 5. The tRNA pool was isolated from yeast and human total RNA as described 79 . Native tRNAs of interest were affinity purified from total tRNA using biotinylated oligonucleotide probes (IDT) immobilized to a streptavidin sepharose (Cytiva) solid support 80 .…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Profiling of tRNA Using an Unexplored Reverse Transcriptase with High Processivity

Nakano,

Gamper,

McGuigan

et al. 2023

Preprint

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Monitoring the dynamic changes of cellular tRNA pools is challenging, due to the extensive post-transcriptional modifications of individual species. The most critical component in tRNAseq is a processive reverse transcriptase (RT) that can read through each modification with high efficiency. Here we show that the recently developed group-II intron RT Induro has the processivity and efficiency necessary to profile tRNA dynamics. Using our Induro-tRNAseq, simpler and more comprehensive than the best methods to date, we show that Induro progressively increases readthrough of tRNA over time and that the mechanism of increase is selective removal of RT stops, without altering the misincorporation frequency. We provide a parallel dataset of the misincorporation profile of Induro relative to the related TGIRT RT to facilitate the prediction of non-annotated modifications. We report an unexpected modification profile among human proline isoacceptors, absent from mouse and lower eukaryotes, that indicates new biology of decoding proline codons.

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Purification and Use of tRNA for Enzymatic Post-translational Addition of Amino Acids to Proteins

Cited by 14 publications

References 17 publications

Phenotypic screening of covalent compound libraries identifies chloromethyl ketone antibiotics and MiaA as a new target

Phenotypic screening of covalent compound libraries identifies chloromethyl ketone antibiotics and MiaA as a new target

Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium

Genome-Wide Profiling of tRNA Using an Unexplored Reverse Transcriptase with High Processivity

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