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Current Gene Therapy 2016The tetracycline-controlled Tet-Off and Tet-On gene expression systems are used to regulate the activity of genes in eukaryotic cells in diverse settings, varying from... (Review)
Review
The tetracycline-controlled Tet-Off and Tet-On gene expression systems are used to regulate the activity of genes in eukaryotic cells in diverse settings, varying from basic biological research to biotechnology and gene therapy applications. These systems are based on regulatory elements that control the activity of the tetracycline-resistance operon in bacteria. The Tet-Off system allows silencing of gene expression by administration of tetracycline (Tc) or tetracycline-derivatives like doxycycline (dox), whereas the Tet-On system allows activation of gene expression by dox. Since the initial design and construction of the original Tet-system, these bacterium-derived systems have been significantly improved for their function in eukaryotic cells. We here review how a dox-controlled HIV-1 variant was designed and used to greatly improve the activity and dox-sensitivity of the rtTA transcriptional activator component of the Tet-On system. These optimized rtTA variants require less dox for activation, which will reduce side effects and allow gene control in tissues where a relatively low dox level can be reached, such as the brain.
Topics: AIDS Vaccines; Bacterial Proteins; Carrier Proteins; Doxycycline; Escherichia coli; Gene Expression Regulation; Genetic Therapy; HIV-1; Humans; Operon; Promoter Regions, Genetic; Tetracycline; Transfection; Vaccines, Attenuated
PubMed: 27216914
DOI: 10.2174/1566523216666160524144041 -
Cell Reports Dec 2020Many bacteria contain an RNA repair operon, encoding the RtcB RNA ligase and the RtcA RNA cyclase, that is regulated by the RtcR transcriptional activator. Although RtcR...
Many bacteria contain an RNA repair operon, encoding the RtcB RNA ligase and the RtcA RNA cyclase, that is regulated by the RtcR transcriptional activator. Although RtcR contains a divergent version of the CARF (CRISPR-associated Rossman fold) oligonucleotide-binding regulatory domain, both the specific signal that regulates operon expression and the substrates of the encoded enzymes are unknown. We report that tRNA fragments activate operon expression. Using a genetic screen in Salmonella enterica serovar Typhimurium, we find that the operon is expressed in the presence of mutations that cause tRNA fragments to accumulate. RtcA, which converts RNA phosphate ends to 2', 3'-cyclic phosphate, is also required. Operon expression and tRNA fragment accumulation also occur upon DNA damage. The CARF domain binds 5' tRNA fragments ending in cyclic phosphate, and RtcR oligomerizes upon binding these ligands, a prerequisite for operon activation. Our studies reveal a signaling pathway involving broken tRNAs and implicate the operon in tRNA repair.
Topics: Humans; Operon; RNA; RNA, Transfer
PubMed: 33357439
DOI: 10.1016/j.celrep.2020.108527 -
Cold Spring Harbor Perspectives in... Jan 2016The cytoplasm of prokaryotes contains many molecular machines interacting directly with the chromosome. These vital interactions depend on the chromosome structure, as a... (Review)
Review
The cytoplasm of prokaryotes contains many molecular machines interacting directly with the chromosome. These vital interactions depend on the chromosome structure, as a molecule, and on the genome organization, as a unit of genetic information. Strong selection for the organization of the genetic elements implicated in these interactions drives replicon ploidy, gene distribution, operon conservation, and the formation of replication-associated traits. The genomes of prokaryotes are also very plastic with high rates of horizontal gene transfer and gene loss. The evolutionary conflicts between plasticity and organization lead to the formation of regions with high genetic diversity whose impact on chromosome structure is poorly understood. Prokaryotic genomes are remarkable documents of natural history because they carry the imprint of all of these selective and mutational forces. Their study allows a better understanding of molecular mechanisms, their impact on microbial evolution, and how they can be tinkered in synthetic biology.
Topics: Biological Evolution; Chromosome Segregation; Chromosomes; DNA Replication; Escherichia coli; Gene Transfer, Horizontal; Genome; Genome Size; Models, Genetic; Operon; Ploidies; Prokaryotic Cells; Recombination, Genetic
PubMed: 26729648
DOI: 10.1101/cshperspect.a018168 -
Gut Microbes 2024Comensal () and are often linked to gut inflammation. However, the causes for variability of pro-inflammatory surface antigens that affect gut commensal/opportunistic...
Comensal () and are often linked to gut inflammation. However, the causes for variability of pro-inflammatory surface antigens that affect gut commensal/opportunistic dualism in remain unclear. By using the classical lipopolysaccharide/O-antigen ' operon' in as a surface antigen model (5-gene-cluster ), and a recent typing strategy for strain classification, we characterized the integrity and conservancy of the entire operon in . Through exploratory analysis of complete genomes and metagenomes, we discovered that most have the operon fragmented into nonrandom patterns of gene-singlets and doublets/triplets, termed 'gene-clusters', or rfb-'minioperons' if predicted as transcriptional. To reflect global operon integrity, contiguity, duplication, and fragmentation principles, we propose a six-category (infra/supra-numerary) cataloging system and a Global Operon Profiling System for bacteria. Mechanistically, genomic sequence analyses revealed that operon fragmentation is driven by intra-operon insertions of predominantly -DNA () and likely natural selection in gut-wall specific micro-niches or micropathologies. -insertions, also detected in other antigenic operons (fimbriae), but not in operons deemed essential (ribosomal), could explain why have fewer KEGG-pathways despite large genomes. DNA insertions, overrepresenting DNA-exchange-avid () species, impact our interpretation of functional metagenomics data by inflating by inflating gene-based pathway inference and by overestimating 'extra-species' abundance. Of disease relevance, species isolated from cavitating/cavernous fistulous tract (CavFT) microlesions in Crohn's Disease have supra-numerary fragmented operons, stimulate TNF-alpha from macrophages with low potency, and do not induce hyperacute peritonitis in mice compared to CavFT . The impact of 'foreign-DNA' insertions on pro-inflammatory operons, metagenomics, and commensalism/opportunism requires further studies to elucidate their potential for novel diagnostics and therapeutics, and to elucidate the role of co-existing pathobionts in Crohn's disease microlesions.
Topics: Operon; Mice; Gastrointestinal Microbiome; Animals; Humans; Metagenomics; Crohn Disease; Bacteroidetes; Antigens, Bacterial; Genome, Bacterial; Enterobacteriaceae
PubMed: 38841888
DOI: 10.1080/19490976.2024.2350150 -
Molekuliarnaia Biologiia 2018The origin of bioluminescence in living organisms was first mentioned by Charles Darwin (1859) and remains obscure despite significant success achieved over the past... (Review)
Review
The origin of bioluminescence in living organisms was first mentioned by Charles Darwin (1859) and remains obscure despite significant success achieved over the past decades. Here we discuss the mechanisms of bacterial bioluminescence. We have the main results from structural and functional analysis of the genes of lux operons, enzymes (luciferase), and mechanisms of bioluminescence in several species of marine bacteria, which belong to three genera, Vibrio, Aliivibrio, and Photobacterium (A. fischeri, V. harveyi, P. leiognathi, and P. phosphoreum), and in terrestrial bacteria of the genus Photorhabdus (Ph. luminescens). The structure and mechanisms for the regulation of the expression of the lux operons are discussed. The fundamental characteristics of luciferase and luciferase-catalyzed reactions (stages of FMNH2 and tetradecanal oxidation, dimensional structure, as well as folding and refolding of the macromolecule) are described. We also discuss the main concepts of the origin of bacterial bioluminescence and its role in the ecology of modern marine fauna, including its involvement in the processes of detoxification of the reactive oxygen species and DNA repair, as well as the bait hypothesis.
Topics: Aliivibrio; Bacterial Proteins; DNA, Bacterial; Genes, Bacterial; Luciferases; Luminescence; Operon; Photobacterium; Vibrio
PubMed: 30633237
DOI: 10.1134/S0026898418060186 -
WormBook : the Online Review of C.... Apr 2015Nearly 15% of the ~20,000 C. elegans genes are contained in operons, multigene clusters controlled by a single promoter. The vast majority of these are of a type where... (Review)
Review
Nearly 15% of the ~20,000 C. elegans genes are contained in operons, multigene clusters controlled by a single promoter. The vast majority of these are of a type where the genes in the cluster are ~100 bp apart and the pre-mRNA is processed by 3' end formation accompanied by trans-splicing. A spliced leader, SL2, is specialized for operon processing. Here we summarize current knowledge on several variations on this theme including: (1) hybrid operons, which have additional promoters between genes; (2) operons with exceptionally long (> 1 kb) intercistronic regions; (3) operons with a second 3' end formation site close to the trans-splice site; (4) alternative operons, in which the exons are sometimes spliced as a single gene and sometimes as two genes; (5) SL1-type operons, which use SL1 instead of SL2 to trans-splice and in which there is no intercistronic space; (6) operons that make dicistronic mRNAs; and (7) non-operon gene clusters, in which either two genes use a single exon as the 3' end of one and the 5' end of the next, or the 3' UTR of one gene serves as the outron of the next. Each of these variations is relatively infrequent, but together they show a remarkable variety of tight-linkage gene arrangements in the C. elegans genome.
Topics: Animals; Caenorhabditis elegans; Genes, Helminth; Multigene Family; Operon
PubMed: 25936768
DOI: 10.1895/wormbook.1.175.1 -
MSystems Dec 2022Anti-CRISPR (Acr) proteins are encoded by (pro)viruses to inhibit their host's CRISPR-Cas systems. Genes encoding Acr and Aca (Acr associated) proteins often colocalize...
Anti-CRISPR (Acr) proteins are encoded by (pro)viruses to inhibit their host's CRISPR-Cas systems. Genes encoding Acr and Aca (Acr associated) proteins often colocalize to form operons. Here, we present AcaFinder as the first Aca genome mining tool. AcaFinder can (i) predict Acas and their associated operons using guilt-by-association (GBA); (ii) identify homologs of known Acas using an HMM (Hidden Markov model) database; (iii) take input genomes for potential prophages, CRISPR-Cas systems, and self-targeting spacers (STSs); and (iv) provide a standalone program (https://github.com/boweny920/AcaFinder) and a web server (http://aca.unl.edu/Aca). AcaFinder was applied to mining over 16,000 prokaryotic and 142,000 gut phage genomes. After a multistep filtering, 36 high-confident new Aca families were identified, which is three times that of the 12 known Aca families. Seven new Aca families were from major human gut bacteria (, , and ) and their phages, while most known Aca families were from and . A complex association network between Acrs and Acas was revealed by analyzing their operonic colocalizations. It appears very common in evolution that the same genes can recombine with different genes and to form diverse operon combinations. At least four bioinformatics programs have been published for genome mining of Acrs since 2020. In contrast, no bioinformatics tools are available for automated Aca discovery. As the self-transcriptional repressor of operons, Aca can be viewed as anti-anti-CRISPRs, with great potential in the improvement of CRISPR-Cas technology. Although all the 12 known Aca proteins contain a conserved helix-turn-helix (HTH) domain, not all HTH-containing proteins are Acas. However, HTH-containing proteins with adjacent Acr homologs encoded in the same genetic operon are likely Aca proteins. AcaFinder implements this guilt-by-association idea and the idea of using HMMs of known Acas for homologs into one software package. Applying AcaFinder in screening prokaryotic and gut phage genomes reveals a complex operonic colocalization network between different families of Acrs and Acas.
Topics: Humans; CRISPR-Cas Systems; Bacteria; Bacteriophages; Operon; Prophages
PubMed: 36413017
DOI: 10.1128/msystems.00817-22 -
RNA (New York, N.Y.) May 2023Transfer RNA fragments are proposed to regulate numerous processes in eukaryotes, including translation inhibition, epigenetic inheritance, and cancer. In the bacterium...
Transfer RNA fragments are proposed to regulate numerous processes in eukaryotes, including translation inhibition, epigenetic inheritance, and cancer. In the bacterium serovar Typhimurium, 5' tRNA halves ending in 2',3' cyclic phosphate are proposed to bind the RtcR transcriptional activator, resulting in transcription of an RNA repair operon. However, since 5' and 3' tRNA halves can remain base paired after cleavage, the 5' tRNA halves could potentially bind RtcR as nicked tRNAs. Here we report that nicked tRNAs are ligands for RtcR. By isolating RNA from bacteria under conditions that preserve base pairing, we show that many tRNA halves are in the form of nicked tRNAs. Using a circularly permuted tRNA that mimics a nicked tRNA, we show that nicked tRNA ending in 2',3' cyclic phosphate is a better ligand for RtcR than the corresponding 5' tRNA half. In human cells, we show that some tRNA halves similarly remain base paired as nicked tRNAs following cleavage by anticodon nucleases. Our work supports a role for the RNA repair operon in repairing nicked tRNAs and has implications for the functions proposed for tRNA fragments in eukaryotes.
Topics: Humans; RNA, Transfer; RNA; Eukaryota; Transcription Factors; Operon; Anticodon
PubMed: 36781286
DOI: 10.1261/rna.079575.122 -
Frontiers in Cellular and Infection... 2019Streptococci, including the dental pathogen , undergo cell-to-cell signaling that is mediated by small peptides to control critical physiological functions such as... (Review)
Review
Streptococci, including the dental pathogen , undergo cell-to-cell signaling that is mediated by small peptides to control critical physiological functions such as adaptation to the environment, control of subpopulation behaviors and regulation of virulence factors. One such model pathway is the regulation of genetic competence, controlled by the ComRS signaling system and the peptide XIP. However, recent research in the characterization of this pathway has uncovered novel operons and peptides that are intertwined into its regulation. These discoveries, such as cell lysis playing a critical role in XIP release and importance of bacterial self-sensing during the signaling process, have caused us to reevaluate previous paradigms and shift our views on the true purpose of these signaling systems. The finding of new peptides such as the ComRS inhibitor XrpA and the peptides of the RcrRPQ operon also suggests there may be more peptides hidden in the genomes of streptococci that could play critical roles in the physiology of these organisms. In this review, we summarize the recent findings in regarding the integration of other circuits into the ComRS signaling pathway, the true mode of XIP export, and how the RcrRPQ operon controls competence activation. We also look at how new technologies can be used to re-annotate the genome to find new open reading frames that encode peptide signals. Together, this summary of research will allow us to reconsider how we perceive these systems to behave and lead us to expand our vocabulary of peptide signals within the genus .
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genome, Bacterial; Operon; Peptides; Quorum Sensing; Signal Transduction; Streptococcus mutans
PubMed: 31245303
DOI: 10.3389/fcimb.2019.00194 -
PeerJ 2022Cyanobacteria are important participants in global biogeochemical process, but their metabolic processes and genomic functions are incompletely understood. In...
Cyanobacteria are important participants in global biogeochemical process, but their metabolic processes and genomic functions are incompletely understood. In particular, operon structure, which can provide valuable metabolic and genomic insight, is difficult to determine experimentally, and algorithmic operon predictions probably underestimate actual operon extent. A software method is presented for enhancing current operon predictions by incorporating information from whole-genome time-series expression studies, using a Machine Learning classifier. Results are presented for the marine cyanobacterium . A total of 15 operon enhancements are proposed. The source code is publicly available.
Topics: Humans; Nitrogen Fixation; Cyanobacteria; Operon; Gene Expression
PubMed: 35433132
DOI: 10.7717/peerj.13259