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Debapratim Dutta, Ivan A Belashov, Joseph E Wedekind
Non-coding RNAs engage in numerous biological activities including gene regulation. To fully understand RNA function it is necessary to probe biologically relevant conformations in living cells. To address this challenge, we coupled RNA-mediated regulation of the GFPuv-reporter gene to icSHAPE (<u>i</u>n <u>c</u>ell <u><u>S</u></u>elective 2´-<u>H</u>ydroxyl <u><u>A</u></u>cylation analyzed by <u><u>P</u></u>rimer <u>E</u>xtension)...
June 13, 2018: Biochemistry
Miroslav Krepl, Jennifer Vögele, Holger Kruse, Elke Duchardt-Ferner, Jens Wöhnert, Jiri Sponer
The neomycin sensing riboswitch is the smallest biologically functional RNA riboswitch, forming a hairpin capped with a U-turn loop-a well-known RNA motif containing a conserved uracil. It was shown previously that a U→C substitution of the eponymous conserved uracil does not alter the riboswitch structure due to C protonation at N3. Furthermore, cytosine is evolutionary permitted to replace uracil in other U-turns. Here, we use molecular dynamics simulations to study the molecular basis of this substitution in the neomycin sensing riboswitch and show that a structure-stabilizing monovalent cation-binding site in the wild-type RNA is the main reason for its negligible structural effect...
June 9, 2018: Nucleic Acids Research
Madhan R Tirumalai, Victor G Stepanov, Andrea Wünsche, Saied Montazari, Racquel O Gonzalez, Kasturi Venkateswaran, George E Fox
BACKGROUND: Bacillus strains producing highly resistant spores have been isolated from cleanrooms and space craft assembly facilities. Organisms that can survive such conditions merit planetary protection concern and if that resistance can be transferred to other organisms, a health concern too. To further efforts to understand these resistances, the complete genome of Bacillus safensis strain FO-36b, which produces spores resistant to peroxide and radiation was determined. The genome was compared to the complete genome of B...
June 8, 2018: BMC Microbiology
Shubin Li, Tao Sun, Chunxiao Xu, Lei Chen, Weiwen Zhang
The fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 2973) has been considered a good chassis candidate for "microbial cell factory" as it can perform oxygenic photosynthesis and its doubling time can be as short as 1.9hours. However, the limited genetic tools currently restrict its further research and application efforts using synthetic biology approaches. In this study, a series of genetic tools were systematically developed and optimized for Synechococcus 2973...
June 5, 2018: Metabolic Engineering
Andrew John Knappenberger, Caroline Wetherington Reiss, Scott A Strobel
Two classes of riboswitches related to the ykkC guanidine-I riboswitch bind phosphoribosyl pyrophosphate (PRPP) and guanosine tetraphosphate (ppGpp). Here we report the co-crystal structure of the PRPP aptamer and its ligand. We also report the structure of the G96A point mutant that prefers ppGpp over PRPP with a dramatic 40,000-fold switch in specificity. The ends of the aptamer form a helix that is not present in the guanidine aptamer and is involved in the expression platform. In the mutant, the base of ppGpp replaces G96 in three-dimensional space...
June 7, 2018: ELife
Paul D Donovan, Linda M Holland, Lisa Lombardi, Aisling Y Coughlan, Desmond G Higgins, Kenneth H Wolfe, Geraldine Butler
Riboswitches are non-coding RNA molecules that regulate gene expression by binding to specific ligands. They are primarily found in bacteria. However, one riboswitch type, the thiamin pyrophosphate (TPP) riboswitch, has also been described in some plants, marine protists and fungi. We find that riboswitches are widespread in the budding yeasts (Saccharomycotina), and they are most common in homologs of DUR31, originally described as a spermidine transporter. We show that DUR31 (an ortholog of N. crassa gene NCU01977) encodes a thiamin transporter in Candida species...
May 31, 2018: PLoS Genetics
V Dhamodharan, Yoko Nomura, Mohammed Dwidar, Yohei Yokobayashi
Optical control of biomolecules via engineered proteins or photoactive small molecules has had a profound impact on biology. However, optochemical tools to control RNA functions in living cells are relatively limited. We synthesized a photoactivatable (photocaged) guanine to modulate gene expression under riboswitch control in both mammalian cells and Escherichia coli by light.
May 30, 2018: Chemical Communications: Chem Comm
Ronald R Breaker
A growing collection of bacterial riboswitch classes is being discovered that sense central metabolites, coenzymes, and signaling molecules. Included among the various mechanisms of gene regulation exploited by these RNA regulatory elements are several that modulate messenger RNA (mRNA) translation. In this review, the mechanisms of riboswitch-mediated translation control are summarized to highlight both their diversity and potential ancient origins. These mechanisms include ligand-gated presentation or occlusion of ribosome-binding sites, control of alternative splicing of mRNAs, and the regulation of mRNA stability...
May 29, 2018: Cold Spring Harbor Perspectives in Biology
Sharrol T Bachas, Adrian R Ferré-D'Amaré
The large yybP-ykoY family of bacterial riboswitches is broadly distributed phylogenetically. Previously, these gene-regulatory RNAs were proposed to respond to Mn2+ . X-ray crystallography revealed a binuclear cation-binding pocket. This comprises one hexacoordinate site, with six oxygen ligands, which preorganizes the second, with five oxygen and one nitrogen ligands. The relatively soft nitrogen ligand was proposed to confer affinity for Mn2+ , but how this excludes other soft cations remained enigmatic...
May 4, 2018: Cell Chemical Biology
Aaron DeLoughery, Jean-Benoît Lalanne, Richard Losick, Gene-Wei Li
Endonucleolytic cleavage within polycistronic mRNAs can lead to differential stability, and thus discordant abundance, among cotranscribed genes. RNase Y, the major endonuclease for mRNA decay in Bacillus subtilis , was originally identified for its cleavage activity toward the cggR-gapA operon, an event that differentiates the synthesis of a glycolytic enzyme from its transcriptional regulator. A three-protein Y-complex (YlbF, YmcA, and YaaT) was recently identified as also being required for this cleavage in vivo, raising the possibility that it is an accessory factor acting to regulate RNase Y...
May 24, 2018: Proceedings of the National Academy of Sciences of the United States of America
Daan C Swarts, Martin Jinek
Cas9 and Cas12a are multidomain CRISPR-associated nucleases that can be programmed with a guide RNA to bind and cleave complementary DNA targets. The guide RNA sequence can be varied, making these effector enzymes versatile tools for genome editing and gene regulation applications. While Cas9 is currently the best-characterized and most widely used nuclease for such purposes, Cas12a (previously named Cpf1) has recently emerged as an alternative for Cas9. Cas9 and Cas12a have distinct evolutionary origins and exhibit different structural architectures, resulting in distinct molecular mechanisms...
May 22, 2018: Wiley Interdisciplinary Reviews. RNA
Madeline E Sherlock, Narasimhan Sudarsan, Ronald R Breaker
Riboswitches are noncoding portions of certain mRNAs that bind metabolite, coenzyme, signaling molecule, or inorganic ion ligands and regulate gene expression. Most known riboswitches sense derivatives of RNA monomers. This bias in ligand chemical composition is consistent with the hypothesis that widespread riboswitch classes first emerged during the RNA World, which is proposed to have existed before proteins were present. Here we report the discovery and biochemical validation of a natural riboswitch class that selectively binds guanosine tetraphosphate (ppGpp), a widespread signaling molecule and bacterial "alarmone" derived from the ribonucleotide GTP...
May 21, 2018: Proceedings of the National Academy of Sciences of the United States of America
Svetlana V Harbaugh, Jennifer Martin, Jenna Weinstein, Grant Ingram, Nancy Kelley-Loughnane
Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes...
May 17, 2018: Methods: a Companion to Methods in Enzymology
Cristopher Segovia, Raul Arias-Carrasco, Alejandro J Yañez, Vinicius Maracaja-Coutinho, Javier Santander
Piscirickettsia salmonis, a fastidious Gram-negative intracellular facultative bacterium, is the causative agent o Piscirickettsiosis. P. salmonis has broad host range with a nearly worldwide distribution, causing significant mortality. The molecular regulatory mechanisms of P. salmonis pathogenesis are relatively unknown, mainly due to its difficult in vitro culture and genomic differences between genogroups. Bacterial non-coding RNAs (ncRNAs) are important post-transcriptional regulators of bacterial physiology and virulence that are predominantly transcribed from intergenic regions (trans-acting) or antisense strand of open reading frames (cis-acting)...
2018: PloS One
Krishna C Suddala, Javier Cabello-Villegas, Malgorzata Michnicka, Collin Marshall, Edward P Nikonowicz, Nils G Walter
In Gram-positive bacteria, T-box riboswitches control gene expression to maintain the cellular pools of aminoacylated tRNAs essential for protein biosynthesis. Co-transcriptional binding of an uncharged tRNA to the riboswitch stabilizes an antiterminator, allowing transcription read-through, whereas an aminoacylated tRNA does not. Recent structural studies have resolved two contact points between tRNA and Stem-I in the 5' half of the T-box riboswitch, but little is known about the mechanism empowering transcriptional control by a small, distal aminoacyl modification...
May 14, 2018: Nature Communications
John C H Spence
Recent advances in the application of X-ray lasers to structural biology are providing time-resolved high-resolution imaging of many processes, from enzyme kinetics to the riboswitch in action, drug action, and light-sensitive proteins.
May 1, 2018: IUCrJ
Guohui Xiao, Liangliang Kong, Rongbo Che, Yusi Yi, Qinchao Zhang, Jie Yan, Xu'ai Lin
Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira species. The most common species, Leptospira interrogans , can transfer from contaminated soil or water to the human body. It is able to survive these changing environments through sensing and responding to the changes of environmental cues. Cyclic di-GMP (c-di-GMP) is a special secondary messenger in bacteria, which can respond to the environment and regulate diverse bacterial behaviors. The c-di-GMP levels in bacterial cells are regulated by diguanylatecyclases (DGC) and phosphodiesterases (PDE), which are responsible for synthesizing or hydrolyzing c-di-GMP, respectively...
2018: Frontiers in Microbiology
Yingying Cai, Miaomiao Xia, Huina Dong, Yuan Qian, Tongcun Zhang, Beiwei Zhu, Jinchuan Wu, Dawei Zhang
BACKGROUND: As a very important coenzyme in the cell metabolism, Vitamin B12 (cobalamin, VB12 ) has been widely used in food and medicine fields. The complete biosynthesis of VB12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB12 production. High-yield VB12 -producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed. RESULT: By the help of engineered strains with varied capacities of VB12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device...
May 11, 2018: BMC Biotechnology
Carl J Balibar, Artjohn Villafania, Christopher M Barbieri, Nick Murgolo, Terry Roemer, Hao Wang, John A Howe
A riboflavin biosynthesis pathway-specific phenotypic screen using a library of compounds, all with unspecified antibiotic activity, identified one small molecule later named ribocil, for which intrinsic antibacterial activity against Escherichia coli was completely suppressed by addition of exogenous riboflavin to the bacterial growth medium. The ability of riboflavin to suppress the activity of ribocil, and further demonstration that ribocil inhibited riboflavin synthesis (IC50  = 0.3 μM), supported that a component of the riboflavin synthesis pathway was the molecular target...
2018: Methods in Molecular Biology
Michael T Wolfinger, Christoph Flamm, Ivo L Hofacker
Riboswitches form an abundant class of cis-regulatory RNA elements that mediate gene expression by binding a small metabolite. For synthetic biology applications, they are becoming cheap and accessible systems for selectively triggering transcription or translation of downstream genes. Many riboswitches are kinetically controlled, hence knowledge of their co-transcriptional mechanisms is essential. We present here an efficient implementation for analyzing co-transcriptional RNA-ligand interaction dynamics. This approach allows for the first time to model concentration-dependent metabolite binding/unbinding kinetics...
May 3, 2018: Methods: a Companion to Methods in Enzymology
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