Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle.
Gel-filtration experiments indicate that a peptide (P2) composed of the basic region of GCN4 fused to the leucine heptad repeats of Lac repressor forms tetrameric aggregates. Gel-shift experiments were performed to determine the orientation of the helices in the tetrameric P2 aggregate. Sandwich-complex formation of peptide P2 with two DNA fragments containing two symmetrical CRE binding sites (5'-ATGACGTCAT-3') at a distance of 21 bp suggests antiparallel aggregation of the Lac leucine heptad repeats. Thus, we conclude that the leucine heptad repeats of Lac repressor have the ability to form homomeric 4-helical bundles with an antiparallel arrangement of the helices. This topology enables the two DNA fragments in the sandwich complexes to be held together by two tetramers of peptide P2. Replacement of the uncharged amino acids of the helical g and e positions of peptide P2 by the corresponding charged residues of GCN4 (peptide P4) results in a dimeric and parallel aggregation of the leucine heptad repeats, and consequently abolishes the potential to form sandwich structures. Similarly, a hybrid Lac repressor in which the GCN4 leucine zipper replaces the natural Lac leucine heptad repeats forms dimers only. It regains the ability to form tetramers when the charged amino acids in helical positions g and e are replaced by uncharged alanines.
Alberti
S
S
Institut für Genetik, Universität zu Köln, FRG.
Oehler
S
S
von Wilcken-Bergmann
B
B
Müller-Hill
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry; D007930:Leucine / Q000737:chemistry* / Q000235:genetics; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; D007930:Leucine