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Riboswitch AND Sensor

Jin-Ho Lee, Volker F Wendisch
The biotechnological production of amino acids occurs at the million-ton scale and annually about 6milliontons of l-glutamate and l-lysine are produced by Escherichia coli and Corynebacterium glutamicum strains. l-glutamate and l-lysine production from starch hydrolysates and molasses is very efficient and access to alternative carbon sources and new products has been enabled by metabolic engineering. This review focusses on genetic and metabolic engineering of amino acid producing strains. In particular, rational approaches involving modulation of transcriptional regulators, regulons, and attenuators will be discussed...
May 15, 2017: Bioresource Technology
Yu Xiu, Sungho Jang, J Andrew Jones, Nicholas A Zill, Robert J Linhardt, Qipeng Yuan, Gyoo Yeol Jung, Mattheos A G Koffas
The ability to design and construct combinatorial synthetic metabolic pathways has far exceeded our capacity for efficient screening and selection of the resulting microbial strains. The need for high-throughput rapid screening techniques is of upmost importance for the future of synthetic biology and metabolic engineering. Here we describe the development of a RNA riboswitch-based biosensor module with dual fluorescent reporters, and demonstrate a high-throughput flow cytometry-based screening method for identification of naringenin over producing Escherichia coli strains in co-culture...
May 20, 2017: Biotechnology and Bioengineering
Joseph E Wedekind, Debapratim Dutta, Ivan A Belashov, Jermaine L Jenkins
Divalent ions fulfill essential cellular roles and are required for virulence by certain bacteria. Free intracellular Mg(2+) can approach 5 mm, but at this level Mn(2+), Ni(2+), or Co(2+) can be growth-inhibitory, and magnesium fluoride is toxic. To maintain ion homeostasis, many bacteria have evolved ion sensors embedded in the 5'-leader sequences of mRNAs encoding ion uptake or efflux channels. Here, we review current insights into these "metalloriboswitches," emphasizing ion-specific binding by structured RNA aptamers and associated conformational changes in downstream signal sequences...
June 9, 2017: Journal of Biological Chemistry
Michael S Goodson, Annastacia C Bennett, Brenton R Jennewine, Emily Briskin, Svetlana V Harbaugh, Nancy Kelley-Loughnane
If fieldable riboswitch-based biological sensors are to fulfil their potential, it is necessary to increase their signal output. Here we report a novel modular amplification system using a riboswitch to initiate signaling between a sensing strain and a reporter strain of E. coli. A quorum sensing signaling molecule biologically wires the sensing and reporter strains together. The amplification circuit increased the amount of fluorescence generated on ligand binding compared to when the riboswitch controlled fluorescence expression directly...
April 21, 2017: ACS Synthetic Biology
Maja Etzel, Mario Mörl
In synthetic biology, metabolic engineering, and gene therapy, there is a strong demand for orthogonal or externally controlled regulation of gene expression. Here, RNA-based regulatory devices represent a promising emerging alternative to proteins, allowing a fast and direct control of gene expression, as no synthesis of regulatory proteins is required. Besides programmable ribozyme elements controlling mRNA stability, regulatory RNA structures in untranslated regions are highly interesting for engineering approaches...
March 7, 2017: Biochemistry
Svetlana V Harbaugh, Michael S Goodson, Kateri Dillon, Sarah Zabarnick, Nancy Kelley-Loughnane
Riboswitches are RNA-based "sensors" that utilize chemically induced structural changes in the 5'-untranslated region of mRNA to regulate expression of downstream genes. Coupling a specific riboswitch with a reporter gene system translates chemical detection by the cell into a quantifiable reporter protein signal. For the majority of reporter gene systems, the readout signal is only expressed in the presence of the target analyte. This makes it difficult to determine the viability and localization of the uninduced biosensor when it is used for "real-word" applications...
February 9, 2017: ACS Synthetic Biology
Elnaz Mehdizadeh Aghdam, Malte Sinn, Vahideh Tarhriz, Abolfazl Barzegar, Jörg S Hartig, Mohammad Saeid Hejazi
Riboswitches are located in non-coding areas of mRNAs and act as sensors of cellular small molecules, regulating gene expression in response to ligand binding. The TPP riboswitch is the most widespread riboswitch occurring in all three domains of life. However, it has been rarely characterized in environmental bacteria other than Escherichia coli and Bacillus subtilis. In this study, TPP riboswitches located in the 5' UTR of thiC operon from Alishewanella tabrizica and Alishewanella aestuarii were identified and characterized...
January 2017: Microbiological Research
Daniel Álvarez, Björn Voß, Dirk Maass, Florian Wüst, Patrick Schaub, Peter Beyer, Ralf Welsch
Phytoene synthase (PSY) catalyzes the highly regulated, frequently rate-limiting synthesis of the first biosynthetically formed carotene. While PSY constitutes a small gene family in most plant taxa, the Brassicaceae, including Arabidopsis (Arabidopsis thaliana), predominantly possess a single PSY gene. This monogenic situation is compensated by the differential expression of two alternative splice variants (ASV), which differ in length and in the exon/intron retention of their 5'UTRs. ASV1 contains a long 5'UTR (untranslated region) and is involved in developmentally regulated carotenoid formation, such as during deetiolation...
December 2016: Plant Physiology
Regine Hengge
The bacterial second messenger c-di-GMP controls bacterial biofilm formation, motility, cell cycle progression, development and virulence. It is synthesized by diguanylate cyclases (with GGDEF domains), degraded by specific phosphodiesterases (PDEs, with EAL of HD-GYP domains) and sensed by a wide variety of c-di-GMP-binding effectors that control diverse targets. c-di-GMP-binding effectors can be riboswitches as well as proteins with highly diverse structures and functions. The latter include 'degenerate' GGDEF/EAL domain proteins that are enzymatically inactive but still able to bind c-di-GMP...
November 5, 2016: Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences
Tobias Busche, Anika Winkler, Ina Wedderhoff, Christian Rückert, Jörn Kalinowski, Darío Ortiz de Orué Lucana
The secreted protein HbpS, the membrane-embedded sensor kinase SenS and the cytoplasmic response regulator SenR from streptomycetes have been shown to form a novel type of signaling pathway. Based on structural biology as well as different biochemical and biophysical approaches, redox stress-based post-translational modifications in the three proteins were shown to modulate the activity of this signaling pathway. In this study, we show that the homologous system, named here HbpSc-SenSc-SenRc, from the model species Streptomyces coelicolor A3(2) provides this bacterium with an efficient defense mechanism under conditions of oxidative stress...
2016: PloS One
Chelsea M Hull, Philip C Bevilacqua
Pathogens are recognized by the innate immune system in part via their unique and complex RNA signatures. A key sensor in human innate immunity is the RNA-activated protein kinase, protein kinase R (PKR), which has two double-stranded RNA (dsRNA) binding motifs (dsRBMs) at its N-terminus. Early studies described PKR as being activated potently by long stretches of perfect dsRNA, a signature typical of viruses. More recently, we and others have found that PKR is also activated by RNAs having structural defects such as bulges and internal loops...
June 21, 2016: Accounts of Chemical Research
J L Litke, M You, S R Jaffrey
Genetically encoded small-molecule sensors are important tools for revealing the dynamics of metabolites and other small molecules in live cells over time. We recently developed RNA-based sensors that exhibit fluorescence in proportion to a small-molecule ligand. One class of these RNA-based sensors are termed Spinach riboswitches. These are RNAs that are based on naturally occurring riboswitches, but have been fused to the Spinach aptamer. The resulting RNA is a fluorogenic riboswitch, producing fluorescence upon binding the cognate small-molecule analyte...
2016: Methods in Enzymology
Simon Ketterer, Lukas Gladis, Adnan Kozica, Matthias Meier
A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to the actuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integration chip...
July 8, 2016: Nucleic Acids Research
Peter Rugbjerg, Hans Jasper Genee, Kristian Jensen, Kira Sarup-Lytzen, Morten Otto Alexander Sommer
Predictable integration of foreign biological signals and parts remains a key challenge in the systematic engineering of synthetic cellular actuations, and general methods to improve signal transduction and sensitivity are needed. To address this problem we modeled and built a molecular signal buffer network in Saccharomyces cerevisiae inspired by chemical pH buffer systems. The molecular buffer system context-insulates a riboswitch enabling synthetic control of colony formation and modular signal manipulations...
July 15, 2016: ACS Synthetic Biology
Chelsea M Hull, Ananya Anmangandla, Philip C Bevilacqua
The innate immune system provides the first line of defense against pathogens through the recognition of nonspecific patterns in RNA to protect the cell in a generalized way. The human RNA-activated protein kinase, PKR, is a dsRNA binding protein and an essential sensor in the innate immune response, which recognizes viral and bacterial pathogens through their RNAs. Upon activation via RNA-dependent autophosphorylation, PKR phosphorylates the eukaryotic initiation factor eIF2α, leading to termination of translation...
April 15, 2016: ACS Chemical Biology
Saki Inuzuka, Shigeyoshi Matsumura, Yoshiya Ikawa
Cyclic diguanylate (c-di-GMP) is a second messenger of bacteria and its detection is an important issue in basic and applied microbiology. As c-di-GMP riboswitch ligand-binding domains (aptamer domains) capture c-di-GMP with high affinity and selectivity, they are promising platforms for the development of RNA-based c-di-GMP sensors. We analyzed two previously reported c-di-GMP sensor RNAs derived from the Vc2 riboswitch. We also designed and tested their variants, some of which showed improved properties as RNA-based c-di-GMP sensors...
August 2016: Journal of Bioscience and Bioengineering
Genichiro Tsuji, Herman O Sintim
A cyclic dinucleotide riboswitch has been fused with a G-quadruplex motif to produce a conditional riboswitch-peroxidase-mimicking sensor that oxidizes both colorimetric and fluorogenic substrates in the presence of c-di-GMP. We find that signal-to-noise ratio could be improved by using a two-, not three-, floor split G-quadruplex for this conditional peroxidase-mimicking riboswitch.
March 2016: Molecular BioSystems
Amin Espah Borujeni, Dennis M Mishler, Jingzhi Wang, Walker Huso, Howard M Salis
Riboswitches are shape-changing regulatory RNAs that bind chemicals and regulate gene expression, directly coupling sensing to cellular actuation. However, it remains unclear how their sequence controls the physics of riboswitch switching and activation, particularly when changing the ligand-binding aptamer domain. We report the development of a statistical thermodynamic model that predicts the sequence-structure-function relationship for translation-regulating riboswitches that activate gene expression, characterized inside cells and within cell-free transcription-translation assays...
January 8, 2016: Nucleic Acids Research
Chang Ho Lee, Seung Ryul Han, Seong-Wook Lee
Aptamers bind to their targets with high affinity and specificity through structure-based complementarity, instead of sequence complementarity that is used by most of the oligonucleotide-based therapeutics. This property has been exploited in using aptamers as multifunctional therapeutic units, by attaching them to therapeutic drugs, nanoparticles, or imaging agents, or as direct molecular decoys for inducing loss-of-function or gain-of-function of targets. One of the most interesting fields of aptamer application is their development as molecular sensors to regulate artificial riboswitches...
February 2016: Nucleic Acid Therapeutics
Piero Morandini
Modulation of metabolic fluxes in plants is usually not a successful business. The main reason is our limited understanding of metabolic plasticity and metabolic control, with the latter still largely influenced by the idea that each pathway has a rate limiting step controlling the flux. Not only is experimental evidence for such steps lacking for most pathways, despite intensive search, but there are also theoretical arguments against the idea that highly regulated enzymes catalyzing reactions far from equilibrium must be considered a priori rate limiting...
April 2009: Plant Science: An International Journal of Experimental Plant Biology
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