PMID:7499290
| Citation |
Duggan, LJ, Hill, TM, Wu, S, Garrison, K, Zhang, X and Gottlieb, PA (1995) Using modified nucleotides to map the DNA determinants of the Tus-TerB complex, the protein-DNA interaction associated with termination of replication in Escherichia coli. J. Biol. Chem. 270:28049-54 |
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| Abstract |
A series of modified nucleotides was used to map the hydrogen-bonding and hydrophobic sites of the TerB DNA required for Tus interaction. Each of four consensus guanine residues in the TerB-binding site was replaced by 7-deazaguanine, 2-aminopurine, or inosine nucleobase analogues, and each thymine by a uracil analogue. The observable equilibrium dissociation constant for the Tus protein-TerB DNA complex was measured at pH 7.5, 25 degrees C, and 150 mM potassium glutamate using a competition binding method. Substitutions made at position 10 with a 7-deazaguanine, 2-aminopurine, or inosine analogue had a large effect on the stability of the complex, approximately +3 kcal/mol in each case. Substitutions made at positions 13 and 17 had a varied response. For uracil substitutions, potential hydrophobic sites were identified at six positions in the TerB DNA. The energetic penalty for the removal of a single methyl group ranged between +1 and +2 kcal/mol. Rate dissociation measurements agree with these results. Overall, major and minor groove determinants are required for binding. An unusual result was that the conserved nucleotide at position 6 did not significantly affect in vitro binding of the complex. |
| Links | |
| Keywords |
2-Aminopurine; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Base Sequence; Binding Sites; Binding, Competitive; Calorimetry; DNA Replication; DNA, Bacterial/chemistry; DNA, Bacterial/metabolism; DNA-Binding Proteins/metabolism; Deoxyribonucleotides; Escherichia coli Proteins; Genes, Bacterial; Guanine/analogs & derivatives; Hydrogen Bonding; Inosine; Kinetics; Molecular Sequence Data; Oligodeoxyribonucleotides; Structure-Activity Relationship; Terminator Regions, Genetic; Thymine; Uracil/analogs & derivatives |
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