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David B Mckay - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of yeast initiation factor 4a a dead box RNA Helicase
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Jonathan M Caruthers, E R Johnson, David B MckayAbstract:The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA Helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 A resolution; it has a parallel α-β topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related Helicases. Using data to 2.8 A resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a “dumbbell” structure consisting of two compact domains connected by an extended linker. By using the structures of other Helicases as a template, compact structures can be modeled for eIF4A that suggest ( i ) Helicase motif IV binds RNA; ( ii ) Arg-298, which is conserved in the DEA(D/H)-box RNA Helicase family but is absent from many other Helicases, also binds RNA; and ( iii ) motifs V and VI “link” the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding.
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Crystal structure of yeast initiation factor 4A, a DEAD-box RNA Helicase
Proceedings of the National Academy of Sciences, 2000Co-Authors: Jonathan M Caruthers, E R Johnson, David B MckayAbstract:The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA Helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 A resolution; it has a parallel alpha-beta topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related Helicases. Using data to 2.8 A resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a "dumbbell" structure consisting of two compact domains connected by an extended linker. By using the structures of other Helicases as a template, compact structures can be modeled for eIF4A that suggest (i) Helicase motif IV binds RNA; (ii) Arg-298, which is conserved in the DEA(D/H)-box RNA Helicase family but is absent from many other Helicases, also binds RNA; and (iii) motifs V and VI "link" the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding.
Radhakrishnan Padmanabhan - One of the best experts on this subject based on the ideXlab platform.
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modulation of the nucleoside triphosphatase RNA Helicase and 5 RNA triphosphatase activities of dengue virus type 2 nonstructural protein 3 ns3 by interaction with ns5 the RNA dependent RNA polymerase
Journal of Biological Chemistry, 2005Co-Authors: Tadahisa Teramoto, Niklaus H Mueller, Jessica Phelan, Vannakambadi K Ganesh, Krishna H M Murthy, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA Helicase and a 5′-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA Helicase are presented. The NS3FL showed an enhanced RNA Helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5′-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA Helicase and RTPase activities of NS3.
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Modulation of the Nucleoside Triphosphatase/RNA Helicase and 5′-RNA Triphosphatase Activities of Dengue Virus Type 2 Nonstructural Protein 3 (NS3) by Interaction with NS5, the RNA-dependent RNA Polymerase
Journal of Biological Chemistry, 2005Co-Authors: Tadahisa Teramoto, Niklaus H Mueller, Jessica Phelan, Vannakambadi K Ganesh, Krishna H M Murthy, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA Helicase and a 5′-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA Helicase are presented. The NS3FL showed an enhanced RNA Helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5′-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA Helicase and RTPase activities of NS3.
Joonho Choe - One of the best experts on this subject based on the ideXlab platform.
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RNA stimulated atpase and RNA Helicase activities and RNA binding domain of hepatitis g virus nonstructural protein 3
Journal of Virology, 1999Co-Authors: Yousang Gwack, Inyoung Song, Joonho ChoeAbstract:Hepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA Helicase proteins. In order to examine the RNA Helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed in Escherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA Helicase activities. Characterization of the ATPase and RNA Helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the other Flaviviridae NS3 proteins. The HGV NS3 Helicase unwinds RNA-RNA duplexes in a 3′-to-5′ direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/NS3 Helicase bound to RNA, RNA/DNA, and DNA duplexes with 5′ and 3′ overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRTGRGRSGR. Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain.
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Mutational analysis of the hepatitis C virus RNA Helicase.
Journal of Virology, 1997Co-Authors: Yousang Gwack, Joonho ChoeAbstract:The carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA Helicase activity, typical of members of the DEAD box family of RNA Helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. A serine in the X position of the DEXH motif disrupted the NTPase and RNA Helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed.
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RNA Helicase activity of Escherichia coli SecA protein.
Biochemical and Biophysical Research Communications, 1997Co-Authors: Su Kyung Park, Joonho ChoeAbstract:Abstract SecA protein ofEscherichia coli(E. coli), an ATPase essential for the translocation of precursor proteins, was found to have an additional activity of RNA Helicase. This RNA unwinding activity of SecA was tested with two kinds of RNA duplex with different predicted stability. Each of these duplexes is consisted of two strands of unequal length with single-stranded ends. The RNA Helicase activity of SecA required ATP and divalent cations. Confirmation of this activity came from the inhibition of unwinding of the RNA duplex when SecA was preincubated with its own polyclonal antibody. The biological significance of the RNA Helicase activity ofE. coliSecA protein is discussed.
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c terminal domain of the hepatitis c virus ns3 protein contains an RNA Helicase activity
Biochemical and Biophysical Research Communications, 1995Co-Authors: Yousang Gwack, Joonho ChoeAbstract:The Hepatitis C Virus (HCV) NS3 protein contains amino acid motifs of a serine proteinase, a nucleotide triphosphatase (NTPase), and an RNA Helicase based on amino acid sequence analysis. Proteinase and NTPase activities of the HCV NS3 protein were reported by several investigators. Here, we show that the recombinant HCV NS3 protein purified from a T7 promoter and His-tag expression system possesses an RNA Helicase activity. The recombinant HCV NS3 protein consists of 466 amino acids from the carboxy terminal of a HCV NS3 open reading frame and 25 additional residues from the vector. The recombinant HCV NS3 protein was purified by metal-binding chromatography. The Helicase activity requires ATP and divalent cations such as Mg2+ and Mn2+. The Helicase activity was abolished by monoclonal antibody specific to the HCV NS3 protein.
William C. Merrick - One of the best experts on this subject based on the ideXlab platform.
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Found in translation - the discovery of the first RNA Helicase, eIF4A
2020Co-Authors: William C. MerrickAbstract:More than 25 years have passed since the first RNA Helicase was identified. When we were starting to look at this protein – the eukaryotic initiation factor 4A (eIF4A) – as an ATP-driven RNA unwinder – an RNA Helicase – we did not imagine that RNA Helicase enzymes would be among the largest enzyme classes. Nor did we anticipate how widespread and essential these proteins would be for RNA metabolism. But we are certainly pleased that the significance of RNA Helicases is becoming ever more obvious, as gene regulation at the RNA level is beginning to enjoy more of the limelight usually reserved for DNA-related processes. This book, the first volume specifically dedicated to RNA Helicases, provides an impressive testimony to how far we have come from the discovery of eIF4A. Detailed mechanistic studies on several enzymes have been reported, several dozen crystal structures of RNA Helicases have been solved, and a rapidly increasing body of data describes their cellular functions. Yet, despite the impressive journey from the humble beginnings of the eIF4A story to this book, many RNA Helicases still cling to their secrets for specificity and mechanism of action. In this sense, these enzymes hold as much sway today as they did right after we ‘‘stumbled’’ across eIF4A.
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biochemical and kinetic characterization of the RNA Helicase activity of eukaryotic initiation factor 4a
Journal of Biological Chemistry, 1999Co-Authors: George W Rogers, Nancy J Richter, William C. MerrickAbstract:Abstract Eukaryotic initiation factor (eIF) 4A is the prototypic member of the DEAD box family of proteins and has been proposed to act as an RNA Helicase to unwind secondary structure in the 5′-untranslated region of eukaryotic mRNAs. Previous studies have shown that the RNA Helicase activity of eIF4A is dependent on the presence of a second initiation factor, eIF4B. In this report, eIF4A has been demonstrated to function independently of eIF4B as an ATP-dependent RNA Helicase. The biochemical and kinetic properties of this activity were examined. By using a family of RNA duplexes with an unstructured single-stranded region followed by a duplex region of increasing length and stability, it was observed that the initial rate of duplex unwinding decreased with increasing stability of the duplex. Furthermore, the maximum amount of duplex unwound also decreased with increasing stability. Results suggest that eIF4A acts in a non-processive manner. eIF4B and eIF4H were shown to stimulate the Helicase activity of eIF4A, allowing eIF4A to unwind longer, more stable duplexes with both an increase in initial rate and maximum amount of duplex unwound. A simple kinetic model is proposed to explain the mechanism by which eIF4A unwinds RNA duplex structures in an ATP-dependent manner.
Jonathan M Caruthers - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of yeast initiation factor 4a a dead box RNA Helicase
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Jonathan M Caruthers, E R Johnson, David B MckayAbstract:The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA Helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 A resolution; it has a parallel α-β topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related Helicases. Using data to 2.8 A resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a “dumbbell” structure consisting of two compact domains connected by an extended linker. By using the structures of other Helicases as a template, compact structures can be modeled for eIF4A that suggest ( i ) Helicase motif IV binds RNA; ( ii ) Arg-298, which is conserved in the DEA(D/H)-box RNA Helicase family but is absent from many other Helicases, also binds RNA; and ( iii ) motifs V and VI “link” the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding.
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Crystal structure of yeast initiation factor 4A, a DEAD-box RNA Helicase
Proceedings of the National Academy of Sciences, 2000Co-Authors: Jonathan M Caruthers, E R Johnson, David B MckayAbstract:The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA Helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 A resolution; it has a parallel alpha-beta topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related Helicases. Using data to 2.8 A resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a "dumbbell" structure consisting of two compact domains connected by an extended linker. By using the structures of other Helicases as a template, compact structures can be modeled for eIF4A that suggest (i) Helicase motif IV binds RNA; (ii) Arg-298, which is conserved in the DEA(D/H)-box RNA Helicase family but is absent from many other Helicases, also binds RNA; and (iii) motifs V and VI "link" the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding.
