Stereochemical effects of L-tryptophan and its analogues on trp repressor's affinity for operator-DNA.
We have employed a filter binding assay to help study the mechanism by which bound L-tryptophan enables the Escherichia coli trp repressor to bind its operators. We have prepared variants of the trp repressor using structural analogues of the natural corepressor, L-tryptophan, and measured the affinity of these variants for a 20-base pair oligonucleotide duplex containing a symmetrical idealization of the trp operator from the E. coli trpEDCBA operon. By normalizing for each analogue's previously determined affinity for the trp aporepressor, we have estimated the extent to which each of the functional groups of bound L-tryptophan contributes to operator affinity. We discuss the likely role of these functional groups in the context of the crystal structures of the inactive, unliganded trp aporepressor, the liganded, active repressor, an inactive pseudorepressor (Pseudorepressors are formed by analogues of L-tryptophan that bind at the tryptophan-binding site but form near isomorphs of the repressor that have poor affinity for operator-DNA.) and the trp repressor/operator complex. We find that the alpha-amino group and an unsubstituted amino (-NH-) nitrogen of L-tryptophan's indole ring are essential for operator affinity. The former properly orients the corepressor and the latter interacts directly with the DNA. The alpha-carboxyl group, on the other hand, greatly enhances but is not essential for operator binding. The alpha-carboxylate's role, which is dependent on the corepressor's orientation in the binding pocket, is apparently to position the guanidinium group of Arg-84 for favorable contacts with the operator's sugar-phosphate backbone.
Marmorstein
R Q
RQ
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Sigler
P B
PB
D001426:Bacterial Proteins / Q000378:metabolism*; D001482:Base Composition; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D006860:Hydrogen Bonding; D007700:Kinetics; D009841:Oligonucleotides; D009875:Operator Regions, Genetic / Q000187:drug effects*; D011487:Protein Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives / Q000494:pharmacology*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D009841:Oligonucleotides; D012097:Repressor Proteins; D014157:Transcription Factors; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins