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Find video protocols related to scientific articles indexed in Pubmed.
Reprint of: Iron/sulfur proteins biogenesis in prokaryotes: formation, regulation and diversity.
Biochim. Biophys. Acta
PUBLISHED: 05-07-2013
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Iron/sulfur centers are key cofactors of proteins intervening in multiple conserved cellular processes, such as gene expression, DNA repair, RNA modification, central metabolism and respiration. Mechanisms allowing Fe/S centers to be assembled, and inserted into polypeptides have attracted much attention in the last decade, both in eukaryotes and prokaryotes. Basic principles and recent advances in our understanding of the prokaryotic Fe/S biogenesis ISC and SUF systems are reviewed in the present communication. Most studies covered stem from investigations in Escherichia coli and Azotobacter vinelandii. Remarkable insights were brought about by complementary structural, spectroscopic, biochemical and genetic studies. Highlights of the recent years include scaffold mediated assembly of Fe/S cluster, A-type carriers mediated delivery of clusters and regulatory control of Fe/S homeostasis via a set of interconnected genetic regulatory circuits. Also, the importance of Fe/S biosynthesis systems in mediating soft metal toxicity was documented. A brief account of the Fe/S biosynthesis systems diversity as present in current databases is given here. Moreover, Fe/S biosynthesis factors have themselves been the object of molecular tailoring during evolution and some examples are discussed here. An effort was made to provide, based on the E. coli system, a general classification associating a given domain with a given function such as to help next search and annotation of genomes. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.
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Expanding control in bacteria: interplay between small RNAs and transcriptional regulators to control gene expression.
Curr. Opin. Microbiol.
PUBLISHED: 02-13-2013
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Small regulatory RNAs (sRNAs) are now considered as major post-transcriptional regulators of gene expression in bacteria. Their importance is related to their variety in probably all bacterial species as well as to the extreme diversity of physiological functions of their target genes. An increasing amount of data point to an intimate connection between sRNAs and transcriptional regulatory networks to control multiple functions as important as motility or group behavior. The resulting mixed circuits unravel novel regulatory links and their properties are just starting to be characterized.
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Iron/sulfur proteins biogenesis in prokaryotes: formation, regulation and diversity.
Biochim. Biophys. Acta
PUBLISHED: 01-06-2013
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Iron/sulfur centers are key cofactors of proteins intervening in multiple conserved cellular processes, such as gene expression, DNA repair, RNA modification, central metabolism and respiration. Mechanisms allowing Fe/S centers to be assembled, and inserted into polypeptides have attracted much attention in the last decade, both in eukaryotes and prokaryotes. Basic principles and recent advances in our understanding of the prokaryotic Fe/S biogenesis ISC and SUF systems are reviewed in the present communication. Most studies covered stem from investigations in Escherichia coli and Azotobacter vinelandii. Remarkable insights were brought about by complementary structural, spectroscopic, biochemical and genetic studies. Highlights of the recent years include scaffold mediated assembly of Fe/S cluster, A-type carriers mediated delivery of clusters and regulatory control of Fe/S homeostasis via a set of interconnected genetic regulatory circuits. Also, the importance of Fe/S biosynthesis systems in mediating soft metal toxicity was documented. A brief account of the Fe/S biosynthesis systems diversity as present in current databases is given here. Moreover, Fe/S biosynthesis factors have themselves been the object of molecular tailoring during evolution and some examples are discussed here. An effort was made to provide, based on the E. coli system, a general classification associating a given domain with a given function such as to help next search and annotation of genomes. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.
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Positive regulation by small RNAs and the role of Hfq.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-10-2010
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Bacterial small noncoding RNAs carry out both positive and negative regulation of gene expression by pairing with mRNAs; in Escherichia coli, this regulation often requires the RNA chaperone Hfq. Three small regulatory RNAs (sRNAs), DsrA, RprA, and ArcZ, positively regulate translation of the sigma factor RpoS, each pairing with the 5 leader to open up an inhibitory hairpin. In vitro, rpoS interaction with sRNAs depends upon an (AAN)(4) Hfq-binding site upstream of the pairing region. Here we show that both Hfq and this Hfq binding site are required for RprA or ArcZ to act in vivo and to form a stable complex with rpoS mRNA in vitro; both were partially dispensable for DsrA at 37 degrees C. ArcZ sRNA is processed from 121 nt to a stable 56 nt species that contains the pairing region; only the 56 nt ArcZ makes a strong Hfq-dependent complex with rpoS. For each of these sRNAs, the stability of the sRNA*mRNA complexes, rather than their rate of formation, best predicted in vivo activity. These studies demonstrate that binding of Hfq to the rpoS mRNA is critical for sRNA regulation under normal conditions, but if the stability of the sRNA*mRNA complex is sufficiently high, the requirement for Hfq can be bypassed.
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MicA sRNA links the PhoP regulon to cell envelope stress.
Mol. Microbiol.
PUBLISHED: 03-25-2010
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Numerous small RNAs regulators of gene expression exist in bacteria. A large class of them binds to the RNA chaperone Hfq and act by base pairing interactions with their target mRNA, thereby affecting their translation and/or stability. They often have multiple direct targets, some of which may be regulators themselves, and production of a single sRNA can therefore affect the expression of dozens of genes. We show in this study that the synthesis of the Escherichia coli pleiotropic PhoPQ two-component system is repressed by MicA, a sigma(E)-dependent sRNA regulator of porin biogenesis. MicA directly pairs with phoPQ mRNA in the translation initiation region of phoP and presumably inhibits translation by competing with ribosome binding. Consequently, MicA downregulates several members of the PhoPQ regulon. By linking PhoPQ to sigma(E), our findings suggest that major cellular processes such as Mg(2+) transport, virulence, LPS modification or resistance to antimicrobial peptides are modulated in response to envelope stress. In addition, we found that Hfq strongly affects the expression of phoP independently of MicA, raising the possibility that even more sRNAs, which remain to be identified, could regulate PhoPQ synthesis.
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Integrating anaerobic/aerobic sensing and the general stress response through the ArcZ small RNA.
EMBO J.
PUBLISHED: 02-11-2010
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The alternative sigma factor RpoS responds to multiple stresses and activates a large number of genes that allow bacteria to adapt to changing environments. The accumulation of RpoS is regulated at multiple levels, including the regulation of its translation by small regulatory RNAs (sRNAs). A library of plasmids expressing each of 26 Escherichia coli sRNAs that bind Hfq was created to globally and rapidly analyse regulation of an rpoS-lacZ translational fusion. The approach can be easily applied to any gene of interest. When overexpressed, four sRNAs, including OxyS, previously shown to repress rpoS, were observed to repress the expression of the rpoS-lacZ fusion. Along with DsrA and RprA, two previously defined activators of rpoS translation, a third new sRNA activator, ArcZ, was identified. The expression of arcZ is repressed by the aerobic/anaerobic-sensing ArcA-ArcB two-component system under anaerobic conditions and adds translational regulation to the ArcA-ArcB regulon. ArcZ directly represses, and is repressed by, arcB transcription, providing a negative feedback loop that may affect functioning of the ArcA-ArcB regulon.
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Regulating the regulator: an RNA decoy acts as an OFF switch for the regulation of an sRNA.
Genes Dev.
PUBLISHED: 09-03-2009
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Many bacterial small regulatory RNAs (sRNAs) pair with mRNA targets, stimulating or inhibiting mRNA stability and/or translation. Regulation of these sRNAs is usually due to tight transcriptional regulation of synthesis.In this issue of Genes & Development and a related paper in Molecular Microbiology, Figueroa-Bossi and colleagues (pp. 2004-2015) and Overgaard and colleagues report a novel regulatory mechanism in which induction of a competing mRNA acts to titrate away the sRNA, allowing expression of an otherwise strongly inhibited target gene.
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A genetic approach for finding small RNAs regulators of genes of interest identifies RybC as regulating the DpiA/DpiB two-component system.
Mol. Microbiol.
PUBLISHED: 05-12-2009
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In Escherichia coli, the largest class of small regulatory RNAs binds to the RNA chaperone Hfq and regulates the stability and/or translation of specific mRNAs. While recent studies have shown that some mRNAs could be subject to post-transcriptional regulation by sRNAs (e.g. mRNAs found by co-immunoprecipitation with Hfq), no method has yet been described to identify small RNAs that regulate them. We developed a method to easily make translational fusions of genes of interest to the lacZ reporter gene, under the control of a P(BAD)-inducible promoter. A multicopy plasmid library of the E. coli genome can then be used to screen for small RNAs that affect the activity of the fusion. This screening method was first applied to the dpiB gene from the dpiBA operon, which encodes a two-component signal transduction system involved in the SOS response to beta-lactams. One small RNA, RybC, was found to negatively regulate the expression of dpiB. Using mutants in the dpiB-lacZ fusion and compensatory mutations in the RybC sRNA, we demonstrate that RybC directly base pairs with the dpiBA mRNA.
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A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes.
Cell
PUBLISHED: 04-03-2009
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Riboswitches are RNA elements acting in cis, controlling expression of their downstream genes through a metabolite-induced alteration of their secondary structure. Here, we demonstrate that two S-adenosylmethionine (SAM) riboswitches, SreA and SreB, can also function in trans and act as noncoding RNAs in Listeria monocytogenes. SreA and SreB control expression of the virulence regulator PrfA by binding to the 5-untranslated region of its mRNA. Absence of the SAM riboswitches SreA and SreB increases the level of PrfA and virulence gene expression in L. monocytogenes. Thus, the impact of the SAM riboswitches on PrfA expression highlights a link between bacterial virulence and nutrient availability. Together, our results uncover an unexpected role for riboswitches and a distinct class of regulatory noncoding RNAs in bacteria.
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Genetic screens to identify bacterial sRNA regulators.
Methods Mol. Biol.
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Small regulatory RNAs (sRNAs) are versatile regulators that have been shown to be involved in the gene regulation of a growing number of biological pathways in bacteria. While finding the targets of a given sRNA has been the focus of many studies, fewer methods have been described to uncover which, if any, sRNAs regulate a given gene. Here I present two genetic screens that are designed to search for sRNAs regulating a gene of interest. Before the screens are performed, a translational fusion is made between the gene of interest and lacZ, designed so that mostly post-transcriptional effects on the genes expression can be analyzed. I describe here a simple and rapid way to obtain this fusion, even when the transcriptional start site is unknown, by combining PCR or 5RACE with recombination in the chromosome of a special strain of Escherichia coli. The first genetic screen uses a genomic multicopy library to find regulator genes that, when overexpressed, affect the expression of the fusion. While this technique is a classical genetic screen, particular attention is paid to how it can be used to specifically find sRNAs. A second screen is described that takes advantage of a specific library of sRNAs of E. coli that provides an easier and more rapid way to look for sRNA regulation. The library is transformed into the fusion containing strain using a serial transformation protocol developed in microtiter plates. The transformants can then be directly assayed for effects on the beta-galactosidase activity of the fusion in liquid, providing a precise and rapid way to evaluate sRNA regulation. Use of one or both of these screens should help uncover new pathways of regulation by sRNAs.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.