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The Synthesis of the Alternating Copolymer Poly [r(A‐s4U)] by RNA Polymerase of Escherichia coli
Abstract
The transcription of poly [d(A‐T)] by RNA polymerase from Escherichia coli in the presence of ATP and 4‐thiouridine triphosphate (s4UTP) lead to the synthesis of poly[r(A‐s4U)]. The investigation of the polymerisation kinetics furnished the kinetic parameter Km (ATP‐s4UTP) = 0.33 mM and Vmax (ATP‐s4UTP) = 204 nmoles AMP × mg enzyme−1× min−1.
The structure of poly[r(A‐s4U)] was proved by nearest neighbour analysis and calculation of the AMP/s4UMP ratio from the absorption spectrum of poly[r(A‐s4U)]. As shown by sedimentation velocity analysis poly[r(A‐s4U)] exhibited a mean s20, w value of 12 S. Enzymatic hydrolysis and thermal denaturation of poly[r(A‐s4U)] revealed the existence of a large hyperchromicity in the absorption spectrum. The absorption‐temperature profile of poly [r(A‐s4U)] measured at 0.08 M Na+ showe a sharp cooperative transition with a Tm of 52°C.
Citing Literature
Number of times cited: 18
- George L. Kenyon and Judy A. Fee, Probing the Active Sites of Enzymes with Conformationally Restricted Substrate Analogs, Progress in Physical Organic Chemistry, (381-410), (2007).
- Michelle M. Hanna, Suzanne Dissinger, Bradley D. Williams and James E. Colston, Synthesis and characterization of 5-[(4-azidophenacyl)thio]uridine 5'-triphosphate, a cleavable photo-cross-linking nucleotide analog, Biochemistry, 28, 14, (5814), (1989).
- Michelle M. Hanna, [27] Photoaffinity cross-linking methods for studying RNA-protein interactions, RNA Processing Part A: General Methods, 10.1016/0076-6879(89)80113-2, (383-409), (1989).
- Alain FAVRE, Roberto BEZERRA, Eliane HAJNSDORF, Yolande LEMAIGRE DUBREUIL and Alain EXPERT‐BEZANÇON, Substitution of uridine in vivo by the intrinsic photoactivable probe 4‐thiouridine in Escherichia coli RNA, European Journal of Biochemistry, 160, 3, (441-449), (2005).
- Wieslaw J. Smagowicz, Jose V. Castell, Robert M. Clegg and Karl Heinz Scheit, Properties of P3-esters of nucleoside triphosphates as substrates for RNA polymerase from Escherichia coli, Biochemistry, 20, 19, (5538), (1981).
- Hans Richard RACKWITZ and Karl Heinz SCHEIT, The Stereochemical Basis of Template Function, European Journal of Biochemistry, 72, 1, (191-200), (2008).
- C. Janion and K.H. Scheit, The effect of thioketo substitution on uracil-2-aminopurine and uracil-2,6-diaminopurine interactions in polynucleotides, Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 432, 2, (192), (1976).
- Anton STÜTZ and Karl Heinz SCHEIT, Properties of ATP and UTP Analogues with P‐S‐C5′ Bonds, European Journal of Biochemistry, 50, 2, (343-349), (2008).
- Karl Heinz SCHEIT and Peter FAERBER, The Effects of Thioketo Substitution upon Uracil‐Adenine Interactions in Polyribonucleotides, European Journal of Biochemistry, 50, 3, (549-555), (2008).
- R. Vormbrock, R. Morawietz and H.G. Gassen, Codon—Anticodon interaction studied with trinucleoside diphosphates containing 2-thiouridine, 4-thiouridine, 2,4-dithiouridine, or 2-thiocytidine, Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 340, 3, (348), (1974).
- Sunil K. Mazumdar, Wolfram Saenger and Karl Heinz Scheit, Molecular structure of poly-2-thiouridylic acid, a double helix with non-equivalent polynucleotide chains, Journal of Molecular Biology, 85, 2, (213), (1974).
- P. Faerber, The synthesis of the alternating copolymer poly [r(G‐s2C)] by RNA polymerase of E. coli. A polynucleotide with an extremely high thermal stability, FEBS Letters, 44, 1, (111-116), (2001).
- Wolfgang Bähr, Karl‐Heinz Scheit and Peter Faeber, The Effects of Thioketo Substitution upon Uracil‐Adenine Interactions in Polyribonucleotides, European Journal of Biochemistry, 33, 3, (535-544), (2005).
- W. Rohde and A.G. Lezius, Synthesis and properties of an alternating polydeoxynucleotide containing 4-thiodeoxyuridine: Poly[d(A-s4U) · d(A-s4U)], Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 308, 3, (361), (1973).
- A.M. Frischauf and K.H. Scheit, Affinity labeling of E.coli RNA polymerase with substrate and template analogues, Biochemical and Biophysical Research Communications, 53, 4, (1227), (1973).
- Peter Faerber, Reversible blocking of DNA transcription of E. coli DNA dependent RNA polymerase with thiopyrimidine nucleotides, Biochemical and Biophysical Research Communications, 55, 3, (873), (1973).
- Peter Faerber, Karl‐Heinz Scheit and Hans Sommer, A New Polynucleotide Complex Poly(s2C) · poly(I), European Journal of Biochemistry, 27, 1, (109-115), (2005).
- W. Bähr, H. Sommer and K.H. Scheit, Synthesis and properties of oligodeoxy-4-thiothymidylic acid, Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 287, 3, (427), (1972).
