Abstract
DNA sequence coding for the last 121 amino acids of Escherichia coli topoisomerase I was synthesized by PCR and cloned into a plasmid under the control of the T7 promoter. Induction of T7 RNA polymerase in E. coli carrying the plasmid clone resulted in over-expression of this C-terminal domain fragment previously shown to confer higher DNA binding affinity to the enzyme. Purification to homogeneity was achieved by phosphocellulose and single-stranded DNA agaraose chromatography. Direct interaction between this 14K domain and poly(dA) was demonstrated by UV spectroscopy. Noncovalent complexes formed between this fragment and oligo(dT) 8 and oligo(dT) 16 can also be trapped by photo-crosslinking.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Bacteriophage T7
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Base Sequence
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Binding Sites
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Chromatography, Affinity
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Chromatography, Ion Exchange
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Circular Dichroism
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Cloning, Molecular / methods
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DNA / metabolism
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DNA Primers
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DNA Topoisomerases, Type I / biosynthesis*
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DNA Topoisomerases, Type I / metabolism
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DNA-Binding Proteins / biosynthesis*
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DNA-Binding Proteins / metabolism
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Escherichia coli / enzymology*
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Gene Expression
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Kinetics
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Molecular Sequence Data
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Peptide Fragments / biosynthesis*
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Peptide Fragments / isolation & purification
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Peptide Fragments / metabolism
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Poly A / metabolism
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Polymerase Chain Reaction
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Promoter Regions, Genetic
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Protein Conformation
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Spectrophotometry, Ultraviolet
Substances
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DNA Primers
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DNA-Binding Proteins
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Peptide Fragments
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Recombinant Proteins
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Poly A
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poly(dA)
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DNA
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DNA Topoisomerases, Type I