An aromatic stacking interaction between subunits helps mediate DNA sequence specificity: operator site discrimination by phage lambda cI repressor.
Sequence specific DNA binding by regulatory proteins provides the basis for regulation of initiation of transcription. A great deal of progress has been made toward understanding sequence specific recognition by individual protein subunits. An additional level of control that needs to be understood is that due to coupling between the subunits of oligomeric regulatory proteins. An example is the bacteriophage lambda cI repressor, a dimeric protein that regulates the lysogenic to lytic genetic switch of the phage. Two levels of specificity are critical to this regulation. First, like all transcriptional regulators, dimers distinguish operator from nonspecific DNA. Direct readout of the DNA sequence by the recognition helix is considered the well understood mechanism for this. However, differential affinity for O(R)1, O(R)2 and O(R)3 is equally critical to the switch because it mediates opposing regulation of divergent promoters. Site specificity at this second level is less well understood. Conformational adaptation by both the repressor and the different operators appears to be important. To evaluate how subunit-subunit interactions are involved in this process, we investigated the effects on both dimer stability and operator binding of amino acid substitutions at the contacts between the symmetrically related helices-5 in the dimer interface. Substitutions for Tyr88 alter dimer stability and greatly perturb differential operator affinity, but generally do not affect operator versus non-operator specificity. The pattern of these effects suggests that the geometry of the face-to-face aromatic stacking interaction between symmetrically related Tyr88 in each subunit, a group in the dimer interface but far removed from the DNA binding interface, plays a critical role in operator discrimination. Conformational changes in the tertiary structure of the subunits appears to be involved. By contrast, the significant effect of I84S substitution is to greatly decrease affinity for all three operators. Presumably, the altered packing of the dimer interface causes a quarternary structural change that moves the two helix-turn-helix motifs out of register with successive DNA major grooves.
Huang
Y T
YT
Department of Molecular Biology and Biochemistry, University of California, Irvine 92697, USA.
Rusinova
E
E
Ross
J B
JB
Senear
D F
DF
D010582:Bacteriophage lambda / Q000737:chemistry*; D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D005454:Fluorescence Polarization; D008958:Models, Molecular; D009154:Mutation; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D014443:Tyrosine