Probing contacts to the DNA backbone in the trp repressor-operator sequence-specific protein-nucleic acid complex using diastereomeric methylphosphonate analogues.
Fourteen analogue DNA sequences containing the trp operator sequence and a single diastereomeric methylphosphonate linkage are each prepared from the stereochemically pure nucleoside methylphosphonate dimer building block, prepared as a phosphoramidite. The analogue sequences are shown to be single diastereomers on the basis of HPLC analysis of the digestion mixture; in each case, only a single diastereomeric dimer is present. These analogue sequences can be used effectively to probe for interactions to either of the prochiral phosphate oxygens as illustrated by their use to identify critical interactions in the trp repressor-operator complex. In a number of cases, the pairs of diastereomeric analogue sequences exhibit variable binding affinities that can be used to identify one of the prochiral phosphate oxygens as a critical site for complex-stabilizing interactions. Upon the basis of dissociation constants, apparent incremental binding energies are assigned to specific interactions. In all but one example, these identified sites for interactions to the phosphate backbone can be correlated with contacts implicated by the crystal structure analysis of the trp repressor-operator complex.
Smith
S A
SA
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
McLaughlin
L W
LW
D001426:Bacterial Proteins; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D009875:Operator Regions, Genetic; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D013237:Stereoisomerism; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D015335:Molecular Probes; D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins; C032627:methylphosphonic acid