The Experts below are selected from a list of 17448 Experts worldwide ranked by ideXlab platform
Eduardo A Groisman - One of the best experts on this subject based on the ideXlab platform.
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RNA secondary structures regulate three steps of rho dependent transcription termination within a bacterial mRNA leader
Nucleic Acids Research, 2017Co-Authors: Michelle A Kriner, Eduardo A GroismanAbstract:: Transcription termination events in bacteria often require the RNA helicase Rho. Typically, Rho promotes termination at the end of coding sequences, but it can also terminate transcription within leader regions to implement regulatory decisions. Rho-dependent termination requires initial recognition of a Rho utilization (rut) site on a nascent RNA by Rho's primary binding surface. However, it is presently unclear what factors determine the location of transcription termination, how RNA secondary structures influence this process and whether mechanistic differences distinguish constitutive from regulated Rho-dependent terminators. We previously demonstrated that the 5' leader mRNA of the Salmonella corA gene can adopt two mutually exclusive Conformations that dictate accessibility of a rut site to Rho. We now report that the corA leader also controls two subsequent steps of Rho-dependent termination. First, the RNA Conformation that presents an accessible rut site promotes pausing of RNA polymerase (RNAP) at a single Rho-dependent termination site over 100 nt downstream. Second, an additional RNA stem-loop promotes Rho activity and controls the location at which Rho-dependent termination occurs, despite having no effect on initial Rho binding to the corA leader. Thus, the multi-step nature of Rho-dependent termination may facilitate regulation of a given coding region by multiple cytoplasmic signals.
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an RNA motif advances transcription by preventing rho dependent termination
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Anastasia Sevostyanova, Eduardo A GroismanAbstract:The transcription termination factor Rho associates with most nascent bacterial RNAs as they emerge from RNA polymerase. However, pharmacological inhibition of Rho derepresses only a small fraction of these transcripts. What, then, determines the specificity of Rho-dependent transcription termination? We now report the identification of a Rho-antagonizing RNA element (RARE) that hinders Rho-dependent transcription termination. We establish that RARE traps Rho in an inactive complex but does not prevent Rho binding to its recruitment sites. Although translating ribosomes normally block Rho access to an mRNA, inefficient translation of an open reading frame in the leader region of the Salmonella mgtCBR operon actually enables transcription of its associated coding region by favoring an RNA Conformation that sequesters RARE. The discovery of an RNA element that inactivates Rho signifies that the specificity of nucleic-acid binding proteins is defined not only by the sequences that recruit these proteins but also by sequences that antagonize their activity.
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the bacterial transcription termination factor rho coordinates mg2 homeostasis with translational signals
Journal of Molecular Biology, 2015Co-Authors: Michelle A Kriner, Eduardo A GroismanAbstract:The bacterial protein Rho triggers transcription termination at the ends of many operons and when transcription and translation become uncoupled. In addition to these genome wide activities, Rho implements regulation of specific genes by dictating whether RNA polymerase terminates transcription within the 5' leader region or continues into the downstream coding region. Here, we report that the Mg(2+) channel gene corA in Salmonella enterica serovar Typhimurium, which was previously thought to be constitutively expressed, is regulated by a Rho-dependent terminator located within its 5' leader region. We demonstrate that the unusually long and highly conserved corA leader mRNA can adopt two mutually exclusive Conformations that determine whether or not Rho interacts with a Rho utilization site on the nascent RNA and thereby prevents transcription of the corA coding region. The RNA Conformation that promotes Rho-dependent termination is favored by efficient translation of corL, a short open reading frame located within the corA leader. Thus, corA transcription is inversely coupled to corL translation. This mechanism resembles those governing expression of Salmonella's other two Mg(2+) transport genes, suggesting that Rho links Mg(2+) uptake to translational signals.
Alexander Mcpherson - One of the best experts on this subject based on the ideXlab platform.
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satellite tobacco mosaic virus RNA structure and implications for assembly
Current Opinion in Structural Biology, 2001Co-Authors: Steven B Larson, Alexander McphersonAbstract:The initial appearance of 45% of the single-stranded RNA of satellite tobacco mosaic virus in electron density maps suggested the entire RNA Conformation could be delineated. Subsequent work has localized nearly 80% of the RNA as stem-loop elements. Connection of the stem-loops in the most efficient manner produces a persuasive model for the encapsidated RNA. This arrangement has significant implications for virus assembly and for the essential role of RNA.
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refined structure of satellite tobacco mosaic virus at 1 8 a resolution
Journal of Molecular Biology, 1998Co-Authors: Steven B Larson, John Day, Aaron Greenwood, Alexander McphersonAbstract:Abstract The molecular structure of satellite tobacco mosaic virus (STMV) has been refined to 1.8 A resolution using X-ray diffraction data collected from crystals grown in microgravity. The final R value was 0.179 and R free was 0.184 for 219,086 independent reflections. The final model of the asymmetric unit contained amino acid residues 13 to 159 of a coat protein monomer, 21 nucleotides, a sulfate ion, and 168 water molecules. The nucleotides were visualized as 30 helical segments of nine base-pairs with an additional base stacked at each 3′ end, plus a “free” nucleotide, not belonging to the helical segments, but firmly bound by the protein. Sulfate ions are located exactly on 5-fold axes and each is coordinated by ten asparagine side-chains. Of the 10,080 structural waters, 168 per asymmetric unit, about 20% serve to bridge the macromolecular components at protein-protein and protein-nucleic acid interfaces. Binding of RNA to the protein involves some salt linkages, particularly to the phosphate of the free nucleotide, but the major contribution is from an intricate network of hydrogen bonds. There are numerous water molecules in the RNA-protein interface, many serving as intermediate hydrogen bond bridges. The sugar-phosphate backbone contributes most of the donors and acceptors for the RNA. The helical RNA Conformation is nearest that of A form DNA. The central region of a helical segment is most extensively involved in contacts with protein, and exhibits low thermal parameters which increase dramatically toward the ends. The visible RNA represents approximately 59% of the total nucleic acid in the virion and is derived from the single-stranded genome, which has folded upon itself to form helical segments. Linking of the helices and the free nucleotides in a contiguous and efficient manner severely restricts the disposition of the remaining, unseen nucleic acid. Using the remaining nucleotides it is possible to fold the RNA according to motifs that provide a periodic distribution of RNA structural elements compatible with the icosahedrally symmetrical arrangement seen in the crystallographic structure. The intimate relationship between protein and nucleic acid in STMV suggests an assembly pathway based on the cooperative and coordinated co-condensation of RNA with capsid protein dimers.
Michael Laughrea - One of the best experts on this subject based on the ideXlab platform.
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hiv 1 viral RNA is selected in the form of monomers that dimerize in a three step protease dependent process the dis of stem loop 1 initiates viral RNA dimerization
Journal of Molecular Biology, 2007Co-Authors: Rujun Song, Jafar Kafaie, Long Yang, Michael LaughreaAbstract:Abstract We have characterized the viral RNA Conformation in wild-type, protease-inactive (PR – ) and SL1-defective (ΔDIS) human immunodeficiency virus type 1 (HIV-1), as a function of the age of the viruses, from newly released to grown-up (≥24 h old). We report evidence for packaging HIV-1 genomic RNA (gRNA) in the form of monomers in PR – virions, viral RNA rearrangement (not maturation) within PR – HIV-1, protease-dependent formation of thermolabile dimeric viral RNAs, a new form of immature gRNA dimer at about 5 h post virion release, and slow-acting dimerization signals in SL1-defective viruses. The rates of gRNA dimer formation were ≥3-fold and ≥10-fold slower in ΔDIS and PR – viruses than in wild-type, respectively. Thus, the DIS, i.e. the palindrome in the apical loop of SL1, is a dimerization initiation signal, but its role can be masked by one or several slow-acting dimerization site(s) when grown-up SL1-inactive virions are investigated. Grown-up PR – virions are not flawless models for immature virions because gRNA dimerization increases with the age of PR – virions, indicating that the PR – mutation does not “freeze” gRNA Conformation in a nascent primordial state. Our study is the first on gRNA Conformation in newly released mutant or primate retroviruses. It shows for the first time that the packaged retroviral gRNA matures in more than one step, and that formation of immature dimeric viral RNA requires viral protein maturation. The monomeric viral RNAs isolated from budding HIV-1, as modeled by newly released PR – virions, may be seen as dimers that are much more fragile than thermolabile dimers.
Steven B Larson - One of the best experts on this subject based on the ideXlab platform.
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satellite tobacco mosaic virus RNA structure and implications for assembly
Current Opinion in Structural Biology, 2001Co-Authors: Steven B Larson, Alexander McphersonAbstract:The initial appearance of 45% of the single-stranded RNA of satellite tobacco mosaic virus in electron density maps suggested the entire RNA Conformation could be delineated. Subsequent work has localized nearly 80% of the RNA as stem-loop elements. Connection of the stem-loops in the most efficient manner produces a persuasive model for the encapsidated RNA. This arrangement has significant implications for virus assembly and for the essential role of RNA.
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refined structure of satellite tobacco mosaic virus at 1 8 a resolution
Journal of Molecular Biology, 1998Co-Authors: Steven B Larson, John Day, Aaron Greenwood, Alexander McphersonAbstract:Abstract The molecular structure of satellite tobacco mosaic virus (STMV) has been refined to 1.8 A resolution using X-ray diffraction data collected from crystals grown in microgravity. The final R value was 0.179 and R free was 0.184 for 219,086 independent reflections. The final model of the asymmetric unit contained amino acid residues 13 to 159 of a coat protein monomer, 21 nucleotides, a sulfate ion, and 168 water molecules. The nucleotides were visualized as 30 helical segments of nine base-pairs with an additional base stacked at each 3′ end, plus a “free” nucleotide, not belonging to the helical segments, but firmly bound by the protein. Sulfate ions are located exactly on 5-fold axes and each is coordinated by ten asparagine side-chains. Of the 10,080 structural waters, 168 per asymmetric unit, about 20% serve to bridge the macromolecular components at protein-protein and protein-nucleic acid interfaces. Binding of RNA to the protein involves some salt linkages, particularly to the phosphate of the free nucleotide, but the major contribution is from an intricate network of hydrogen bonds. There are numerous water molecules in the RNA-protein interface, many serving as intermediate hydrogen bond bridges. The sugar-phosphate backbone contributes most of the donors and acceptors for the RNA. The helical RNA Conformation is nearest that of A form DNA. The central region of a helical segment is most extensively involved in contacts with protein, and exhibits low thermal parameters which increase dramatically toward the ends. The visible RNA represents approximately 59% of the total nucleic acid in the virion and is derived from the single-stranded genome, which has folded upon itself to form helical segments. Linking of the helices and the free nucleotides in a contiguous and efficient manner severely restricts the disposition of the remaining, unseen nucleic acid. Using the remaining nucleotides it is possible to fold the RNA according to motifs that provide a periodic distribution of RNA structural elements compatible with the icosahedrally symmetrical arrangement seen in the crystallographic structure. The intimate relationship between protein and nucleic acid in STMV suggests an assembly pathway based on the cooperative and coordinated co-condensation of RNA with capsid protein dimers.
Dev P Arya - One of the best experts on this subject based on the ideXlab platform.
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click dimers to target hiv tar RNA Conformation
Biochemistry, 2012Co-Authors: Sunil Kumar, Patrick Kellish, Edward W. Robinson, Deyun Wang, Daniel H. Appella, Dev P AryaAbstract:A series of neomycin dimers have been synthesized using "click chemistry" with varying functionality and length in the linker region to target the human immunodeficiency virus type 1 (HIV-1) TAR RNA region of the HIV virus. The TAR (Trans-Activation Responsive) RNA region, a 59 bp stem-loop structure located at the 5'-end of all nascent viral transcripts, interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1. Neomycin, an aminosugar, has been shown to exhibit multiple binding sites on TAR RNA. This observation prompted us to design and synthesize a library of triazole-linked neomycin dimers using click chemistry. The binding between neomycin dimers and TAR RNA was characterized using spectroscopic techniques, including FID (fluorescent intercalator displacement), a FRET (fluorescence resonance energy transfer) competitive assay, circular dichroism (CD), and UV thermal denaturation. UV thermal denaturation studies demonstrate that binding of neomycin dimers increases the melting temperature (T(m)) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement (FID) and a FRET competition assay revealed nanomolar binding affinity between neomycin dimers and HIV TAR RNA, while in case of neomycin, only weak binding was detected. More importantly, most of the dimers exhibited lower IC(50) values toward HIV TAR RNA, when compared to the fluorescent Tat peptide, and show increased selectivity over mutant TAR RNA. Cytopathic effects investigated using MT-2 cells indicate a number of the dimers with high affinity toward TAR show promising anti-HIV activity.
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Click Dimers To Target HIV TAR RNA Conformation
2012Co-Authors: Sunil Kumar, Patrick Kellish, Edward W. Robinson, Deyun Wang, Daniel H. Appella, Dev P AryaAbstract:A series of neomycin dimers have been synthesized using “click chemistry” with varying functionality and length in the linker region to target the human immunodeficiency virus type 1 (HIV-1) TAR RNA region of the HIV virus. The TAR (Trans-Activation Responsive) RNA region, a 59 bp stem–loop structure located at the 5′-end of all nascent viral transcripts, interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1. Neomycin, an aminosugar, has been shown to exhibit multiple binding sites on TAR RNA. This observation prompted us to design and synthesize a library of triazole-linked neomycin dimers using click chemistry. The binding between neomycin dimers and TAR RNA was characterized using spectroscopic techniques, including FID (fluorescent intercalator displacement), a FRET (fluorescence resonance energy transfer) competitive assay, circular dichroism (CD), and UV thermal denaturation. UV thermal denaturation studies demonstrate that binding of neomycin dimers increases the melting temperature (Tm) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement (FID) and a FRET competition assay revealed nanomolar binding affinity between neomycin dimers and HIV TAR RNA, while in case of neomycin, only weak binding was detected. More importantly, most of the dimers exhibited lower IC50 values toward HIV TAR RNA, when compared to the fluorescent Tat peptide, and show increased selectivity over mutant TAR RNA. Cytopathic effects investigated using MT-2 cells indicate a number of the dimers with high affinity toward TAR show promising anti-HIV activity
