Nucleic Acids Research最新文献

英文中文

Structural and bioinformatics analyses identify deoxydinucleotide-specific nucleases and their association with genomic islands in gram-positive bacteria

IF 14.9 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research

Pub Date : 2025-01-08 DOI: 10.1093/nar/gkae1235

Sofia Mortensen, Stanislava Kuncová, Justin D Lormand, Tanner M Myers, Soo-Kyoung Kim, Vincent T Lee, Wade C Winkler, Holger Sondermann

Dinucleases of the DEDD superfamily, such as oligoribonuclease, Rexo2 and nanoRNase C, catalyze the essential final step of RNA degradation, the conversion of di- to mononucleotides. The active sites of these enzymes are optimized for substrates that are two nucleotides long, and do not discriminate between RNA and DNA. Here, we identified a novel DEDD subfamily, members of which function as dedicated deoxydinucleases (diDNases) that specifically hydrolyze single-stranded DNA dinucleotides in a sequence-independent manner. Crystal structures of enzyme-substrate complexes reveal that specificity for DNA stems from a combination of conserved structural elements that exclude diribonucleotides as substrates. Consistently, diDNases fail to complement the loss of enzymes that act on diribonucleotides, indicating that these two groups of enzymes support distinct cellular functions. The genes encoding diDNases are found predominantly in genomic islands of Actinomycetes and Clostridia, which, together with their association with phage-defense systems, suggest potential roles in bacterial immunity.

{"title":"Structural and bioinformatics analyses identify deoxydinucleotide-specific nucleases and their association with genomic islands in gram-positive bacteria","authors":"Sofia Mortensen, Stanislava Kuncová, Justin D Lormand, Tanner M Myers, Soo-Kyoung Kim, Vincent T Lee, Wade C Winkler, Holger Sondermann","doi":"10.1093/nar/gkae1235","DOIUrl":"https://doi.org/10.1093/nar/gkae1235","url":null,"abstract":"Dinucleases of the DEDD superfamily, such as oligoribonuclease, Rexo2 and nanoRNase C, catalyze the essential final step of RNA degradation, the conversion of di- to mononucleotides. The active sites of these enzymes are optimized for substrates that are two nucleotides long, and do not discriminate between RNA and DNA. Here, we identified a novel DEDD subfamily, members of which function as dedicated deoxydinucleases (diDNases) that specifically hydrolyze single-stranded DNA dinucleotides in a sequence-independent manner. Crystal structures of enzyme-substrate complexes reveal that specificity for DNA stems from a combination of conserved structural elements that exclude diribonucleotides as substrates. Consistently, diDNases fail to complement the loss of enzymes that act on diribonucleotides, indicating that these two groups of enzymes support distinct cellular functions. The genes encoding diDNases are found predominantly in genomic islands of Actinomycetes and Clostridia, which, together with their association with phage-defense systems, suggest potential roles in bacterial immunity.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"22 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142935932","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Molecular mechanisms underlying allosteric behavior of Escherichia coli DgoR, a GntR/FadR family transcriptional regulator

IF 14.9 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research

Pub Date : 2025-01-07 DOI: 10.1093/nar/gkae1299

Swati Singh, Garima Arya, Rajesh Mishra, Shivam Singla, Akhil Pratap, Krishna Upadhayay, Monika Sharma, Rachna Chaba

GntR/FadR family featuring an N-terminal winged helix–turn–helix DNA-binding domain and a C-terminal α-helical effector-binding and oligomerization domain constitutes one of the largest families of transcriptional regulators. Several GntR/FadR regulators govern the metabolism of sugar acids, carbon sources implicated in bacterial–host interactions. Although effectors are known for a few sugar acid regulators, the unavailability of relevant structures has left their allosteric mechanism unexplored. Here, using DgoR, a transcriptional repressor of d-galactonate metabolism in Escherichia coli, as a model, and its superrepressor alleles, we probed allostery in a GntR/FadR family sugar acid regulator. Genetic and biochemical studies established compromised response to d-galactonate as the reason for the superrepressor behavior of the mutants: T180I does not bind d-galactonate, and while A97V, S171L and M188I bind d-galactonate, effector binding does not induce a conformational change required for derepression, suggesting altered allostery. For mechanistic insights into allosteric communication, we performed simulations of the modeled DgoR structure in different allosteric states for both the wild-type and mutant proteins. We found that each mutant exhibits unique dynamics disrupting the intrinsic allosteric communication pathways, thereby impacting DgoR function. We finally validated the allosteric communication model by testing in silico predictions with experimental data.

{"title":"Molecular mechanisms underlying allosteric behavior of Escherichia coli DgoR, a GntR/FadR family transcriptional regulator","authors":"Swati Singh, Garima Arya, Rajesh Mishra, Shivam Singla, Akhil Pratap, Krishna Upadhayay, Monika Sharma, Rachna Chaba","doi":"10.1093/nar/gkae1299","DOIUrl":"https://doi.org/10.1093/nar/gkae1299","url":null,"abstract":"GntR/FadR family featuring an N-terminal winged helix–turn–helix DNA-binding domain and a C-terminal α-helical effector-binding and oligomerization domain constitutes one of the largest families of transcriptional regulators. Several GntR/FadR regulators govern the metabolism of sugar acids, carbon sources implicated in bacterial–host interactions. Although effectors are known for a few sugar acid regulators, the unavailability of relevant structures has left their allosteric mechanism unexplored. Here, using DgoR, a transcriptional repressor of d-galactonate metabolism in Escherichia coli, as a model, and its superrepressor alleles, we probed allostery in a GntR/FadR family sugar acid regulator. Genetic and biochemical studies established compromised response to d-galactonate as the reason for the superrepressor behavior of the mutants: T180I does not bind d-galactonate, and while A97V, S171L and M188I bind d-galactonate, effector binding does not induce a conformational change required for derepression, suggesting altered allostery. For mechanistic insights into allosteric communication, we performed simulations of the modeled DgoR structure in different allosteric states for both the wild-type and mutant proteins. We found that each mutant exhibits unique dynamics disrupting the intrinsic allosteric communication pathways, thereby impacting DgoR function. We finally validated the allosteric communication model by testing in silico predictions with experimental data.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"28 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142935960","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Discovery of highly active kynureninases for cancer immunotherapy through protein language model

IF 14.9 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research

Pub Date : 2025-01-07 DOI: 10.1093/nar/gkae1245

Hyunuk Eom, Sukhwan Park, Kye Soo Cho, Jihyeon Lee, Hyunbin Kim, Stephanie Kim, Jinsol Yang, Young-Hyun Han, Juyong Lee, Chaok Seok, Myeong Sup Lee, Woon Ju Song, Martin Steinegger

Tailor-made enzymes empower a wide range of versatile applications, although searching for the desirable enzymes often requires high throughput screening and thus poses significant challenges. In this study, we employed homology searches and protein language models to discover and prioritize enzymes by their kinetic parameters. We aimed to discover kynureninases as a potentially versatile therapeutic enzyme, which hydrolyses L-kynurenine, a potent immunosuppressive metabolite, to overcome the immunosuppressive tumor microenvironment in anticancer therapy. Subsequently, we experimentally validated the efficacy of four top-ranked kynureninases under in vitro and in vivo conditions. Our findings revealed a catalytically most active one with a nearly twofold increase in turnover number over the prior best and a 3.4-fold reduction in tumor weight in mouse model comparisons. Consequently, our approach holds promise for the targeted quantitative enzyme discovery and selection suitable for specific applications with higher accuracy, significantly broadening the scope of enzyme utilization. A web-executable version of our workflow is available at seekrank.steineggerlab.com and our code is available as free open-source software at github.com/steineggerlab/SeekRank.

{"title":"Discovery of highly active kynureninases for cancer immunotherapy through protein language model","authors":"Hyunuk Eom, Sukhwan Park, Kye Soo Cho, Jihyeon Lee, Hyunbin Kim, Stephanie Kim, Jinsol Yang, Young-Hyun Han, Juyong Lee, Chaok Seok, Myeong Sup Lee, Woon Ju Song, Martin Steinegger","doi":"10.1093/nar/gkae1245","DOIUrl":"https://doi.org/10.1093/nar/gkae1245","url":null,"abstract":"Tailor-made enzymes empower a wide range of versatile applications, although searching for the desirable enzymes often requires high throughput screening and thus poses significant challenges. In this study, we employed homology searches and protein language models to discover and prioritize enzymes by their kinetic parameters. We aimed to discover kynureninases as a potentially versatile therapeutic enzyme, which hydrolyses L-kynurenine, a potent immunosuppressive metabolite, to overcome the immunosuppressive tumor microenvironment in anticancer therapy. Subsequently, we experimentally validated the efficacy of four top-ranked kynureninases under in vitro and in vivo conditions. Our findings revealed a catalytically most active one with a nearly twofold increase in turnover number over the prior best and a 3.4-fold reduction in tumor weight in mouse model comparisons. Consequently, our approach holds promise for the targeted quantitative enzyme discovery and selection suitable for specific applications with higher accuracy, significantly broadening the scope of enzyme utilization. A web-executable version of our workflow is available at seekrank.steineggerlab.com and our code is available as free open-source software at github.com/steineggerlab/SeekRank.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"33 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142935933","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Pervasive RNA-binding protein enrichment on TAD boundaries regulates TAD organization

IF 14.9 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research

Pub Date : 2025-01-07 DOI: 10.1093/nar/gkae1271

Qiang Sun, Qin Zhou, Yulong Qiao, Xiaona Chen, Hao Sun, Huating Wang

Mammalian genome is hierarchically organized by CTCF and cohesin through loop extrusion mechanism to facilitate the organization of topologically associating domains (TADs). Mounting evidence suggests additional factors/mechanisms exist to orchestrate TAD formation and maintenance. In this study, we investigate the potential role of RNA-binding proteins (RBPs) in TAD organization. By integrated analyses of global RBP binding and 3D genome mapping profiles from both K562 and HepG2 cells, our study unveils the prevalent enrichment of RBPs on TAD boundaries and define boundary-associated RBPs (baRBPs). We found that baRBP binding is correlated with enhanced TAD insulation strength and in a CTCF-independent manner. Moreover, baRBP binding is associated with nascent promoter transcription. Additional experimental testing was performed using RBFox2 as a paradigm. Knockdown of RBFox2 in K562 cells causes mild TAD reorganization. Moreover, RBFox2 enrichment on TAD boundaries is a conserved phenomenon in C2C12 myoblast (MB) cells. RBFox2 is downregulated and its bound boundaries are remodeled during MB differentiation into myotubes. Finally, transcriptional inhibition indeed decreases RBFox2 binding and disrupts TAD boundary insulation. Altogether, our findings demonstrate that RBPs can play an active role in modulating TAD organization through co-transcriptional association and synergistic actions with nascent promoter transcripts.

{"title":"Pervasive RNA-binding protein enrichment on TAD boundaries regulates TAD organization","authors":"Qiang Sun, Qin Zhou, Yulong Qiao, Xiaona Chen, Hao Sun, Huating Wang","doi":"10.1093/nar/gkae1271","DOIUrl":"https://doi.org/10.1093/nar/gkae1271","url":null,"abstract":"Mammalian genome is hierarchically organized by CTCF and cohesin through loop extrusion mechanism to facilitate the organization of topologically associating domains (TADs). Mounting evidence suggests additional factors/mechanisms exist to orchestrate TAD formation and maintenance. In this study, we investigate the potential role of RNA-binding proteins (RBPs) in TAD organization. By integrated analyses of global RBP binding and 3D genome mapping profiles from both K562 and HepG2 cells, our study unveils the prevalent enrichment of RBPs on TAD boundaries and define boundary-associated RBPs (baRBPs). We found that baRBP binding is correlated with enhanced TAD insulation strength and in a CTCF-independent manner. Moreover, baRBP binding is associated with nascent promoter transcription. Additional experimental testing was performed using RBFox2 as a paradigm. Knockdown of RBFox2 in K562 cells causes mild TAD reorganization. Moreover, RBFox2 enrichment on TAD boundaries is a conserved phenomenon in C2C12 myoblast (MB) cells. RBFox2 is downregulated and its bound boundaries are remodeled during MB differentiation into myotubes. Finally, transcriptional inhibition indeed decreases RBFox2 binding and disrupts TAD boundary insulation. Altogether, our findings demonstrate that RBPs can play an active role in modulating TAD organization through co-transcriptional association and synergistic actions with nascent promoter transcripts.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"41 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142935935","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Correction to 'Characterization of an interplay between a Mycobacterium tuberculosis MazF homolog, Rv1495 and its sole DNA topoisomerase I'. 结核分枝杆菌 MazF 同源物 Rv1495 与其唯一的 DNA 拓扑异构酶 I 之间相互作用的特征》的更正。

IF 16.6 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research

Pub Date : 2025-01-07 DOI: 10.1093/nar/gkae1226

引用次数: 0

Instruction-responsive programmable assemblies with DNA origami block pieces.

IF 16.6 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research

Pub Date : 2025-01-07 DOI: 10.1093/nar/gkae1193

Fang Wang, Xiaolong Shi, Xin Chen, Di Deng, Sirui Li, Si Sun, Zheng Kou, Jin Xu, Xiaoli Qiang

DNA nanotechnology has created a wide variety of nanostructures that provide a reliable platform for nanofabrication and DNA computing. However, constructing programmable finite arrays that allow for easy pre-functionalization remains challenge. We aim to create more standardized and controllable DNA origami components, which could be assembled into finite-scale and more diverse superstructures driven by instruction sets. In this work, we designed and implemented DNA origami building block pieces (DOBPs) with eight mutually independent programmable edges and formulated DNA instructions that tailored such components. This system enables DOBPs to be assembled into one or more specific 2D arrays according to the instruction sets. Theoretically, a two-unit system can generate up to 48 distinct DNA arrays. Importantly, experiments results demonstrated that DOBPs are capable of both deterministic and nondeterministic assemblies. Moreover, after examining the effects of different connection strategies and instruction implementations on the yield of the target structures, we assembled more complex 2D arrays, including limited self-assembly arrays such as 'square frames', 'windmills' and 'multiples of 3' long strips. We also demonstrated examples of Boolean logic gates 'AND' and 'XOR' computations based on these assembly arrays. The assembly system provides a model nano-structure for the research on controllable finite self-assembly and offers a more integrated approach for the storage and processing of molecular information.

{"title":"Instruction-responsive programmable assemblies with DNA origami block pieces.","authors":"Fang Wang, Xiaolong Shi, Xin Chen, Di Deng, Sirui Li, Si Sun, Zheng Kou, Jin Xu, Xiaoli Qiang","doi":"10.1093/nar/gkae1193","DOIUrl":"10.1093/nar/gkae1193","url":null,"abstract":"DNA nanotechnology has created a wide variety of nanostructures that provide a reliable platform for nanofabrication and DNA computing. However, constructing programmable finite arrays that allow for easy pre-functionalization remains challenge. We aim to create more standardized and controllable DNA origami components, which could be assembled into finite-scale and more diverse superstructures driven by instruction sets. In this work, we designed and implemented DNA origami building block pieces (DOBPs) with eight mutually independent programmable edges and formulated DNA instructions that tailored such components. This system enables DOBPs to be assembled into one or more specific 2D arrays according to the instruction sets. Theoretically, a two-unit system can generate up to 48 distinct DNA arrays. Importantly, experiments results demonstrated that DOBPs are capable of both deterministic and nondeterministic assemblies. Moreover, after examining the effects of different connection strategies and instruction implementations on the yield of the target structures, we assembled more complex 2D arrays, including limited self-assembly arrays such as 'square frames', 'windmills' and 'multiples of 3' long strips. We also demonstrated examples of Boolean logic gates 'AND' and 'XOR' computations based on these assembly arrays. The assembly system provides a model nano-structure for the research on controllable finite self-assembly and offers a more integrated approach for the storage and processing of molecular information.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":" ","pages":""},"PeriodicalIF":16.6,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11724294/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142854793","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Correction to "Growth-regulated co-occupancy of Mediator and Lsm3 at intronic ribosomal protein genes". 更正 "Mediator 和 Lsm3 在核糖体蛋白内含子基因上的生长调控共占位"。

IF 16.6 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research

Pub Date : 2025-01-07 DOI: 10.1093/nar/gkae1258

引用次数: 0

Editor's Note 'Transcription factor binding sites in the pol geneintragenic regulatory region of HIV-1 are important for virus infectivity'.

IF 16.6 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research

Pub Date : 2025-01-07 DOI: 10.1093/nar/gkae1233

引用次数: 0

Direct delivery of Cas-embedded cytosine base editors as ribonucleoprotein complexes for efficient and accurate editing of clinically relevant targets. 以核糖核蛋白复合物的形式直接传递嵌入式胞嘧啶碱基编辑器，以高效、准确地编辑临床相关靶点。

IF 16.6 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research

Pub Date : 2025-01-07 DOI: 10.1093/nar/gkae1217

Jeong Min Lee, Jing Zeng, Pengpeng Liu, My Anh Nguyen, Diego Suchenski Loustaunau, Daniel E Bauer, Nese Kurt Yilmaz, Scot A Wolfe, Celia A Schiffer

Recently, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBEs have the significant liabilities of off-target and bystander editing activities, partly due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study, we engineered optimized, soluble and stable Cas-embedded CBEs (CE_CBEs) that integrate several recent advances, which were efficiently formulated for direct delivery into cells as ribonucleoprotein (RNP) complexes. Our resulting CE_CBE RNP complexes efficiently target cytosines in TC dinucleotides with minimal off-target or bystander mutations. Delivery of additional uracil glycosylase inhibitor protein in trans further increased C-to-T editing efficiency and target purity in a dose-dependent manner, minimizing indel formation. A single electroporation was sufficient to effectively edit the therapeutically relevant locus BCL11A for sickle cell disease in hematopoietic stem and progenitor cells in a dose-dependent manner without cellular toxicity. Significantly, these CE_CBE RNPs permitted highly efficient editing and engraftment of transplanted cells in mice. Thus, our designed CBE proteins provide promising reagents for RNP-based editing at disease-related sites.

最近，胞嘧啶碱基编辑器（CBEs）已成为一种很有前途的治疗工具，可用于特异性编辑单核苷酸变异和破坏与疾病相关的特定基因。尽管前景广阔，但目前可用的 CBEs 仍存在脱靶和旁观者编辑活性的重大缺陷，部分原因在于它们的传递机制，从而限制了它们的潜在应用。在这项研究中，我们设计了优化的、可溶的、稳定的 Cas-embedded CBEs（CE_CBEs），它整合了多项最新进展，并以核糖核蛋白（RNP）复合物的形式有效地直接递送到细胞中。我们的 CE_CBE RNP 复合物能有效靶向 TC 二核苷酸中的胞嘧啶，且脱靶或旁观者突变极少。反式输送额外的尿嘧啶糖基化酶抑制蛋白进一步提高了C-T编辑效率和靶点纯度，并最大程度地减少了吲哚的形成。单次电穿孔足以以剂量依赖的方式有效编辑造血干细胞和祖细胞中镰状细胞病的治疗相关基因座 BCL11A，而不会对细胞造成毒性。值得注意的是，这些 CE_CBE RNPs 可以在小鼠体内实现高效编辑和移植细胞。因此，我们设计的 CBE 蛋白为在疾病相关部位进行基于 RNP 的编辑提供了前景广阔的试剂。

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引用次数: 0

Defining serine tRNA knockout as a strategy for effective repression of gene expression in organisms with a recoded genome

IF 14.9 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research

Pub Date : 2025-01-07 DOI: 10.1093/nar/gkae1266

Peter J Voorhees, Xinyou Chang, Samuel K Lai

Whole genome codon compression—the reassignment of all instances of a specific codon to synonymous codons—can generate organisms capable of tolerating knockout of otherwise essential transfer RNAs (tRNAs). As a result, such knockout strains enable numerous unique applications, such as high-efficiency production of DNA encoding extremely toxic genes or non-canonical proteins. However, achieving stringent control over protein expression in these organisms remains challenging, particularly with proteins where incomplete repression results in deleterious phenotypes. One platform enjoying increasing popularity utilizes serine TCA codon compression, relying on the prevailing understanding that deletion of tRNASer(UGA) (serT) would render the serine codon compressed organism incapable of translating any genes containing TCA codons. Here, we report evidence that tRNASer(CGA) (serU) can, surprisingly, also decode TCA, thereby precluding complete control over expression of TCA-containing genes in organisms with serT deletion. We then demonstrate the conditions necessary, including the precise modifications to the GRO and codon usage within the transgene, to overcome this interaction and achieve exceptionally stringent control over protein expression. Our findings provide critical insights and corresponding methods for guiding future use of serine codon compression for absolute control over protein expression, as well as a general strategy for optimizing repression via compression of other codons.

{"title":"Defining serine tRNA knockout as a strategy for effective repression of gene expression in organisms with a recoded genome","authors":"Peter J Voorhees, Xinyou Chang, Samuel K Lai","doi":"10.1093/nar/gkae1266","DOIUrl":"https://doi.org/10.1093/nar/gkae1266","url":null,"abstract":"Whole genome codon compression—the reassignment of all instances of a specific codon to synonymous codons—can generate organisms capable of tolerating knockout of otherwise essential transfer RNAs (tRNAs). As a result, such knockout strains enable numerous unique applications, such as high-efficiency production of DNA encoding extremely toxic genes or non-canonical proteins. However, achieving stringent control over protein expression in these organisms remains challenging, particularly with proteins where incomplete repression results in deleterious phenotypes. One platform enjoying increasing popularity utilizes serine TCA codon compression, relying on the prevailing understanding that deletion of tRNASer(UGA) (serT) would render the serine codon compressed organism incapable of translating any genes containing TCA codons. Here, we report evidence that tRNASer(CGA) (serU) can, surprisingly, also decode TCA, thereby precluding complete control over expression of TCA-containing genes in organisms with serT deletion. We then demonstrate the conditions necessary, including the precise modifications to the GRO and codon usage within the transgene, to overcome this interaction and achieve exceptionally stringent control over protein expression. Our findings provide critical insights and corresponding methods for guiding future use of serine codon compression for absolute control over protein expression, as well as a general strategy for optimizing repression via compression of other codons.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"18 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142935963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

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Nucleic Acids Research

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