The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform
Heinz G. Floss - One of the best experts on this subject based on the ideXlab platform.
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Overexpression of the Thiostrepton‐resistance Gene from Streptomyces azureus in Escherichia coli and Characterization of Recognition sites of the 23S rRNA A1067 2′‐methyltransferase in the Guanosine Triphosphatase Center of 23S Ribosomal RNA
European journal of biochemistry, 1994Co-Authors: Andreas Bechthold, Heinz G. FlossAbstract:The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-l-methionine (Km= 0.1 mM) and an RNA Fragment containing nucleotides 1029–1122 of the 23s ribosomal RNA from E. coli (Km= 0.001 mM) were determined. S-Adenosyl-l-homocysteine showed competitive product inhibition (Ki= 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA Fragment with mutations in nucleotides 1051–1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.
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overexpression of the thiostrepton resistance gene from streptomyces azureus in escherichia coli and characterization of recognition sites of the 23s rRNA a1067 2 methyltransferase in the guanosine triphosphatase center of 23s ribosomal RNA
FEBS Journal, 1994Co-Authors: Andreas Bechthold, Heinz G. FlossAbstract:The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-l-methionine (Km= 0.1 mM) and an RNA Fragment containing nucleotides 1029–1122 of the 23s ribosomal RNA from E. coli (Km= 0.001 mM) were determined. S-Adenosyl-l-homocysteine showed competitive product inhibition (Ki= 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA Fragment with mutations in nucleotides 1051–1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.
Martin Pelchat - One of the best experts on this subject based on the ideXlab platform.
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Identification of a binding site for ASF/SF2 on an RNA Fragment derived from the hepatitis delta virus genome.
PloS one, 2013Co-Authors: Dorota Sikora, Dajiang Zhang, Teodora Bojic, Yasnee Beeharry, Ali Tanara, Martin PelchatAbstract:The hepatitis delta virus (HDV) is a small (~1700 nucleotides) RNA pathogen which encodes only one open reading frame. Consequently, HDV is dependent on host proteins to replicate its RNA genome. Recently, we reported that ASF/SF2 binds directly and specifically to an HDV-derived RNA Fragment which has RNA polymerase II promoter activity. Here, we localized the binding site of ASF/SF2 on the HDV RNA Fragment by performing binding experiments using purified recombinant ASF/SF2 combined with deletion analysis and site-directed mutagenesis. In addition, we investigated the requirement of ASF/SF2 for HDV RNA replication using RNAi-mediated knock-down of ASF/SF2 in 293 cells replicating HDV RNA. Overall, our results indicate that ASF/SF2 binds to a purine-rich region distant from both the previously published initiation site of HDV mRNA transcription and binding site of RNAP II, and suggest that this protein is not involved in HDV replication in the cellular system used.
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identification of a binding site for asf sf2 on an RNA Fragment derived from the hepatitis delta virus genome
PLOS ONE, 2013Co-Authors: Dorota Sikora, Dajiang Zhang, Teodora Bojic, Yasnee Beeharry, Ali Tanara, Martin PelchatAbstract:The hepatitis delta virus (HDV) is a small (~1700 nucleotides) RNA pathogen which encodes only one open reading frame. Consequently, HDV is dependent on host proteins to replicate its RNA genome. Recently, we reported that ASF/SF2 binds directly and specifically to an HDV-derived RNA Fragment which has RNA polymerase II promoter activity. Here, we localized the binding site of ASF/SF2 on the HDV RNA Fragment by performing binding experiments using purified recombinant ASF/SF2 combined with deletion analysis and site-directed mutagenesis. In addition, we investigated the requirement of ASF/SF2 for HDV RNA replication using RNAi-mediated knock-down of ASF/SF2 in 293 cells replicating HDV RNA. Overall, our results indicate that ASF/SF2 binds to a purine-rich region distant from both the previously published initiation site of HDV mRNA transcription and binding site of RNAP II, and suggest that this protein is not involved in HDV replication in the cellular system used.
Elizabeth J Grayhack - One of the best experts on this subject based on the ideXlab platform.
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control of translation efficiency in yeast by codon anticodon interactions
RNA, 2010Co-Authors: Daniel P Letzring, Kimberly Dean, Elizabeth J GrayhackAbstract:The choice of synonymous codons used to encode a polypeptide contributes to substantial differences in translation efficiency between genes. However, both the magnitude and the mechanisms of codon-mediated effects are unknown, as neither the effects of individual codons nor the parameters that modulate codon-mediated regulation are understood, particularly in eukaryotes. To explore this problem in Saccharomyces cerevisiae, we performed the first systematic analysis of codon effects on expression. We find that the arginine codon CGA is strongly inhibitory, resulting in progressively and sharply reduced expression with increased CGA codon dosage. CGA-mediated inhibition of expression is primarily due to wobble decoding of CGA, since it is nearly completely suppressed by coexpression of an exact match anticodon-mutated tRNAArg(UCG), and is associated with generation of a smaller RNA Fragment, likely due to endonucleolytic cleavage at a stalled ribosome. Moreover, CGA codon pairs are more effective inhibitors of expression than individual CGA codons. These results directly implicate decoding by the ribosome and interactions at neighboring sites within the ribosome as mediators of codon-specific translation efficiency.
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Control of translation efficiency in yeast by codon–anticodon interactions
RNA, 2010Co-Authors: Daniel P Letzring, Kimberly Dean, Elizabeth J GrayhackAbstract:The choice of synonymous codons used to encode a polypeptide contributes to substantial differences in translation efficiency between genes. However, both the magnitude and the mechanisms of codon-mediated effects are unknown, as neither the effects of individual codons nor the parameters that modulate codon-mediated regulation are understood, particularly in eukaryotes. To explore this problem in Saccharomyces cerevisiae, we performed the first systematic analysis of codon effects on expression. We find that the arginine codon CGA is strongly inhibitory, resulting in progressively and sharply reduced expression with increased CGA codon dosage. CGA-mediated inhibition of expression is primarily due to wobble decoding of CGA, since it is nearly completely suppressed by coexpression of an exact match anticodon-mutated tRNAArg(UCG), and is associated with generation of a smaller RNA Fragment, likely due to endonucleolytic cleavage at a stalled ribosome. Moreover, CGA codon pairs are more effective inhibitors of expression than individual CGA codons. These results directly implicate decoding by the ribosome and interactions at neighboring sites within the ribosome as mediators of codon-specific translation efficiency.
Andreas Bechthold - One of the best experts on this subject based on the ideXlab platform.
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Overexpression of the Thiostrepton‐resistance Gene from Streptomyces azureus in Escherichia coli and Characterization of Recognition sites of the 23S rRNA A1067 2′‐methyltransferase in the Guanosine Triphosphatase Center of 23S Ribosomal RNA
European journal of biochemistry, 1994Co-Authors: Andreas Bechthold, Heinz G. FlossAbstract:The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-l-methionine (Km= 0.1 mM) and an RNA Fragment containing nucleotides 1029–1122 of the 23s ribosomal RNA from E. coli (Km= 0.001 mM) were determined. S-Adenosyl-l-homocysteine showed competitive product inhibition (Ki= 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA Fragment with mutations in nucleotides 1051–1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.
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overexpression of the thiostrepton resistance gene from streptomyces azureus in escherichia coli and characterization of recognition sites of the 23s rRNA a1067 2 methyltransferase in the guanosine triphosphatase center of 23s ribosomal RNA
FEBS Journal, 1994Co-Authors: Andreas Bechthold, Heinz G. FlossAbstract:The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-l-methionine (Km= 0.1 mM) and an RNA Fragment containing nucleotides 1029–1122 of the 23s ribosomal RNA from E. coli (Km= 0.001 mM) were determined. S-Adenosyl-l-homocysteine showed competitive product inhibition (Ki= 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA Fragment with mutations in nucleotides 1051–1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.
Floyd E Romesberg - One of the best experts on this subject based on the ideXlab platform.
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fret characterization of complex conformational changes in a large 16s ribosomal RNA Fragment site specifically labeled using unnatural base pairs
ACS Chemical Biology, 2016Co-Authors: Thomas Lavergne, Rajan Lamichhane, Denis A Malyshev, Zhengtao Li, Lingjun Li, Edit Sperling, James R Williamson, David P Millar, Floyd E RomesbergAbstract:Ribosome assembly has been studied intensively using Forster resonance energy transfer (FRET) with fluorophore-labeled Fragments of RNA produced by chemical synthesis. However, these studies are limited by the size of the accessible RNA Fragments. We have developed a replicable unnatural base pair (UBP) formed between (d)5SICS and (d)MMO2 or (d)NaM, which efficiently directs the transcription of RNA containing unnatural nucleotides. We now report the synthesis and evaluation of several of the corresponding ribotriphosphates bearing linkers that enable the chemoselective attachment of different functionalities. We found that the RNA polymerase from T7 bacteriophage does not incorporate NaM derivatives but does efficiently incorporate 5SICSCO, whose linker enables functional group conjugation via Click chemistry, and when combined with the previously identified MMO2A, whose amine side chains permits conjugation via NHS coupling chemistry, enables site-specific double labeling of transcribed RNA. To study ri...
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fret characterization of complex conformational changes in a large 16s ribosomal RNA Fragment site specifically labeled using unnatural base pairs
ACS Chemical Biology, 2016Co-Authors: Thomas Lavergne, Rajan Lamichhane, Denis A Malyshev, Edit Sperling, James R Williamson, David P Millar, Floyd E RomesbergAbstract:Ribosome assembly has been studied intensively using Forster resonance energy transfer (FRET) with fluorophore-labeled Fragments of RNA produced by chemical synthesis. However, these studies are limited by the size of the accessible RNA Fragments. We have developed a replicable unnatural base pair (UBP) formed between (d)5SICS and (d)MMO2 or (d)NaM, which efficiently directs the transcription of RNA containing unnatural nucleotides. We now report the synthesis and evaluation of several of the corresponding ribotriphosphates bearing linkers that enable the chemoselective attachment of different functionalities. We found that the RNA polymerase from T7 bacteriophage does not incorporate NaM derivatives but does efficiently incorporate 5SICS(CO), whose linker enables functional group conjugation via Click chemistry, and when combined with the previously identified MMO2(A), whose amine side chains permits conjugation via NHS coupling chemistry, enables site-specific double labeling of transcribed RNA. To study ribosome assembly, we transcribed RNA corresponding to a 243-nt Fragment of the central domain of Thermus thermophilus 16S rRNA containing 5SICS(CO) and MMO2(A) at defined locations and then site-specifically attached the fluorophores Cy3 and Cy5. FRET was characterized using single-molecule total inteRNAl reflection fluorescence (smTIRF) microscopy in the presence of various combinations of added ribosomal proteins. We demonstrate that each of the Fragment's two three-helix junctions exist in open and closed states, with the latter favored by sequential protein binding. These results elucidate early and previously uncharacterized folding events underlying ribosome assembly and demonstrate the applicability of UBPs for biochemical, structural, and functional studies of RNAs.
