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Riboswitch OR Riboswitches

Iolanda Ferro, Klaus Liebeton, Zoya Ignatova
Cellular growth crucially depends on protein synthesis and the abundance of translational components. Among them, aminoacyl-tRNAs play a central role in biosynthesis and shape the kinetics of mRNA translation, thus influencing protein production. Here, we used microarray-based approaches to determine the charging levels and tRNA abundance of Bacillus licheniformis. We observed an interesting cross-talk among tRNA expression, charging pattern and growth rate. For a large subset of tRNAs, we found a co-regulated and augmented expression at high growth rate...
September 13, 2017: Journal of Molecular Biology
Sungho Jang, Gyoo Yeol Jung
Riboswitches form a class of genetically-encoded sensor-regulators and are considered as promising tools for monitoring various metabolites. Functional parameters of a riboswitch, like dynamic or operational range, should be optimized before it is implemented in a specific application for monitoring the target molecule efficiently. However, optimization of a riboswitch was not straightforward and required detailed studies owing to its complex sequence-function relationship. Here, we present three approaches for tuning and optimization of functional parameters of a riboswitch using an artificial L-tryptophan riboswitch as an example...
September 11, 2017: Biotechnology and Bioengineering
Johann Peltier, Olga Soutourina
Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important signaling molecule for community behavior control, cell morphogenesis, and virulence in bacteria. In addition to protein effectors, this second messenger binds RNA molecules that act as riboswitches to control target gene expression. In this chapter, we describe a method for experimental validation of the functionality of c-di-GMP-responsive riboswitches and the analysis of c-di-GMP control of target gene expression by qRT-PCR and Northern blot...
2017: Methods in Molecular Biology
Prabhadevi Venkataramani, Zhao-Xun Liang
C-di-GMP has emerged as a prevalent bacterial messenger that controls a multitude of bacterial behaviors. Having access to milligram or gram quantities of c-di-GMP is essential for the biochemical and structural characterization of enzymes and effectors involved in c-di-GMP signaling. Although c-di-GMP can be synthesized using chemical methods, diguanylate cyclases (DGC)-based enzymatic synthesis is the most efficient method of preparing c-di-GMP today. Many DGCs are not suitable for c-di-GMP production because of poor protein stability and the presence of a c-di-GMP-binding inhibitory site (I-site) in most DGCs...
2017: Methods in Molecular Biology
Juan J Quereda, Pascale Cossart
Noncoding RNAs (ncRNAs) regulating virulence have been identified in most pathogens. This review discusses RNA-mediated mechanisms exploited by bacterial pathogens to successfully infect and colonize their hosts. It discusses the most representative RNA-mediated regulatory mechanisms employed by two intracellular [Listeria monocytogenes and Salmonella enterica serovar Typhimurium (S. Typhimurium)] and two extracellular (Vibrio cholerae and Staphylococcus aureus) bacterial pathogens. We review the RNA-mediated regulators (e...
September 8, 2017: Annual Review of Microbiology
Juraj Michálik, Hélène Touzet, Yann Ponty
Motivation: Kinetics is key to understand many phenomena involving RNAs, such as co-transcriptional folding and riboswitches. Exact out-of-equilibrium studies induce extreme computational demands, leading state-of-the-art methods to rely on approximated kinetics landscapes, obtained using sampling strategies that strive to generate the key landmarks of the landscape topology. However, such methods are impeded by a large level of redundancy within sampled sets. Such a redundancy is uninformative, and obfuscates important intermediate states, leading to an incomplete vision of RNA dynamics...
July 15, 2017: Bioinformatics
Sumit Mukherjee, Danny Barash, Supratim Sengupta
Riboswitches are cis-regulatory elements that regulate the expression of genes involved in biosynthesis or transport of a ligand that binds to them. Among the nearly 40 classes of riboswitches discovered so far, three are known to regulate the concentration of biologically encoded amino acids glycine, lysine, and glutamine. While some comparative genomics studies of riboswitches focusing on their gross distribution across different bacterial taxa have been carried out recently, systematic functional annotation and analysis of lysine riboswitches and the genes they regulate are still lacking...
2017: PloS One
Hoang Long Pham, Adison Wong, Niying Chua, Wei Suong Teo, Wen Shan Yew, Matthew Wook Chang
Environmental pH is a fundamental signal continuously directing the metabolism and behavior of living cells. Programming the precise cellular response toward environmental pH is, therefore, crucial for engineering cells for increasingly sophisticated functions. Herein, we engineer a set of riboswitch-based pH-sensing genetic devices to enable the control of gene expression according to differential environmental pH. We next develop a digital pH-sensing system to utilize the analogue-sensing behavior of these devices for high-resolution recording of host cell exposure to discrete external pH levels...
September 4, 2017: Nature Communications
Sven Findeiß, Maja Etzel, Sebastian Will, Mario Mörl, Peter F Stadler
RNA aptamers readily recognize small organic molecules, polypeptides, as well as other nucleic acids in a highly specific manner. Many such aptamers have evolved as parts of regulatory systems in nature. Experimental selection techniques such as SELEX have been very successful in finding artificial aptamers for a wide variety of natural and synthetic ligands. Changes in structure and/or stability of aptamers upon ligand binding can propagate through larger RNA constructs and cause specific structural changes at distal positions...
August 30, 2017: Sensors
Nathalie Mangel, Jared B Fudge, Teresa B Fitzpatrick, Wilhelm Gruissem, Hervé Vanderschuren
Vitamin B1, which consists of the vitamers thiamin and its phosphorylated derivatives, is an essential micronutrient for all living organisms because it is required as a metabolic cofactor in several enzymatic reactions. Genetic diversity of vitamin B1 biosynthesis and accumulation has not been investigated in major crop species other than rice and potato. We analyzed cassava germplasm for accumulation of B1 vitamers. Vitamin B1 content in leaves and roots of 41 cassava accessions showed significant variation between accessions...
June 15, 2017: Journal of Experimental Botany
Clarence Y Cheng, Wipapat Kladwang, Joseph D Yesselman, Rhiju Das
Despite the critical roles RNA structures play in regulating gene expression, sequencing-based methods for experimentally determining RNA base pairs have remained inaccurate. Here, we describe a multidimensional chemical-mapping method called "mutate-and-map read out through next-generation sequencing" (M2-seq) that takes advantage of sparsely mutated nucleotides to induce structural perturbations at partner nucleotides and then detects these events through dimethyl sulfate (DMS) probing and mutational profiling...
August 29, 2017: Proceedings of the National Academy of Sciences of the United States of America
Yuling Liao, Yanrui Ye, Bin Wang, Li Pan
OBJECTIVES: To deregulate the purine operon of the purine biosynthetic pathway and optimize energy generation of the respiratory chain to improve the yield of guanosine in Bacillus amyloliquefaciens XH7. RESULTS: The 5'-untranslated region of the purine operon, which contains the guanine-sensing riboswitch, was disrupted. The native promoter Pw in B. amyloliquefaciens XH7 was replaced by different strong promoters. Among the promoter replacement mutants, XH7purE::P41 gave the highest guanosine yield (16...
August 24, 2017: Biotechnology Letters
Mohammed Dwidar, Yohei Yokobayashi
Bdellovibrio bacteriovorus is a predatory bacterium that feeds on Gram-negative bacteria including a wide range of pathogens and thus has potential applications as a biocontrol agent. Owing to its unique life cycle, however, there are limited tools that enable genetic manipulation of B. bacteriovorus. This work describes our first steps toward engineering the predatory bacterium for practical applications by developing basic genetic parts to control gene expression. Specifically, we evaluated four robust promoters that are active during the attack phase of B...
August 18, 2017: ACS Synthetic Biology
Arnaud Taton, Amy T Ma, Mizuho Ota, Susan Golden, James W Golden
To downregulate gene expression in cyanobacteria, we constructed NOT gate genetic circuits using orthogonal promoters and their cognate repressors regulated translationally by synthetic riboswitches. Four NOT gates were tested and characterized in five cyanobacterial strains using fluorescent reporter-gene assays. In comparison to alternative systems used to downregulate gene expression in cyanobacteria, these NOT gates performed well, reducing YFP reporter expression by 4 to 50-fold. We further evaluated these NOT gates by controlling the expression of the ftsZ gene, which encodes a prokaryotic tubulin homolog that is required for cell division and is essential for Synechococcus elongatus PCC 7942...
August 14, 2017: ACS Synthetic Biology
Marion Kirchner, Kenji Schorpp, Kamyar Hadian, Sabine Schneider
Riboswitches are bacterial RNA elements that regulate gene expression in response to metabolite or ion abundance and are considered as potential drug targets. In recent years a number of methods to find non-natural riboswitch ligands have been described. Here we report a high-throughput in vivo screening system that allows identifying OFF-riboswitch modulators in a 384 well bioluminescence assay format. We use a reverse reporter gene setup in Bacillus subtilis, consisting of a primary screening assay, a secondary assay as well as counter assays to detect compounds in a library of 1,280 molecules that act on the guanine-responsive xpt riboswitch from B...
August 10, 2017: Scientific Reports
Carolyn E Carr, Luis A Marky
Intramolecular three-way junctions are commonly found in both DNA and RNA. These structures are functionally relevant in ribozymes, riboswitches, rRNA, and during replication. In this work, we present a thermodynamic description of the unfolding of DNA intramolecular three-way junctions. We used a combination of spectroscopic and calorimetric techniques to investigate the folding/unfolding thermodynamics of two three-way junctions with a closed (Closed-J) or open (Open-J) junction and their appropriate control stem-loop motifs (GAAATT-Hp, CTATC-Hp, and Dumbbell)...
August 8, 2017: Biophysical Journal
Dmitriy Ignatov, Jörgen Johansson
Bacterial pathogens encounter several different environments during an infection, many of them possibly being detrimental. In order to sense its surroundings and adjust the gene expression accordingly, different regulatory schemes are undertaken. With these, the bacterium appropriately can differentiate between various environmental cues to express the correct virulence factor at the appropriate time and place. An attractive regulator device is RNA, which has an outstanding ability to alter its structure in response to external stimuli, such as metabolite concentration or alterations in temperature, to control its downstream gene expression...
August 9, 2017: Wiley Interdisciplinary Reviews. RNA
I Boeras, B Seufzer, S Brady, A Rendahl, X Heng, K Boris-Lawrie
The paradigm protein synthesis rate is regulated by structural complexity of the 5'untranslated region (UTR) derives from bacterial and other riboswitches. In-solution, HIV-1 5'UTR forms two interchangeable long-range nucleotide (nt) -pairings, one sequesters the gag start codon promoting dimerization while the other sequesters the dimer initiation signal preventing dimerization. While the effect of these nt-pairings on dimerization and packaging has been documented their effect on authentic HIV translation in cellulo has remained elusive until now...
July 31, 2017: Scientific Reports
Xiaoqing Liu, Jian Tian, Lihui Liu, Tao Zhu, Xiaoxia Yu, Xiaoyu Chu, Bin Yao, Ningfeng Wu, Yunliu Fan
Fluorine is ubiquitous and the most active non-metal element in nature. While many microorganisms have developed fluoride resistance as a result of the widespread and prolonged application of oral hygiene products, the mechanisms used by these organisms to overcome fluoride toxicity are incompletely understood. In this study, a fluoride-resistant strain, Enterobacter cloacae FRM, was identified which could grow well at a fluoride concentration of 4,000 mg/L. According to comparative genomics, transcriptome under fluoride stress, and sequence analyses of two fluoride-resistant fosmid clones, the genomic island GI3 was found to be important for fluoride resistance...
July 28, 2017: Scientific Reports
Sungho Jang, Sungyeon Jang, Yu Xiu, Taek Jin Kang, Sang-Hyeup Lee, Mattheos A G Koffas, Gyoo Yeol Jung
Microbial strains are considered promising hosts for production of flavonoids because of their rapid growth rate and suitability for large-scale manufacturing. However, productivity and titer of current recombinant strains still do not meet the requirements of industrial processes. Genetically encoded biosensors have been applied for high-throughput screening or dynamic regulation of biosynthetic pathways for enhancing the performance of microbial strains that produce valuable chemicals. Currently, few protein sensor-regulators for flavonoids exist...
August 10, 2017: ACS Synthetic Biology
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