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.
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.
D015075:2-Aminopurine; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002151:Calorimetry; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003854:Deoxyribonucleotides; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006147:Guanine / Q000031:analogs & derivatives; D006860:Hydrogen Bonding; D007288:Inosine; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D013329:Structure-Activity Relationship; D013728:Terminator Regions, Genetic; D013941:Thymine; D014498:Uracil / Q000031:analogs & derivatives
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D003854:Deoxyribonucleotides; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; C061910:tus protein, Bacteria; C489236:tus protein, E coli; D015075:2-Aminopurine; D014498:Uracil; D007288:Inosine; D006147:Guanine; C066856:7-deazaguanine; D013941:Thymine