DNA-based loss of specificity mutations. Effects of DNA sequence on the contacted and non-contacted base preferences of bacteriophage P22 repressor.
Although the two central bases of the P22 operator are not contacted by the P22 repressor, changes in these bases alter the affinity of operator for repressor. Previous studies (Wu, L., and Koudelka, G. B. (1993) J. Biol. Chem. 268, 18975-18981) show that the structure of the P22 repressor-operator complex varies with central base sequence. Here we show that central base sequence composition affects the strength of two, and likely all, specific amino acid-base pair contacts between synthetic P22 operators and P22 repressor. However, altering a specific protein-DNA contact via a loss-of-contact mutation in repressor results in a loss of specificity at only one contacted position. Thus, only changing the sequence of non-contacted bases affects repressor's global base specificity. The observed effects of ionic concentration on the affinities of various operators for repressor and the DNase I patterns of protein complexes with these binding sites indicate certain central base sequences facilitate optimal juxtaposition of repressor with its contacted bases, while others prevent it. The existence of different structural forms of the repressor-operator complexes explains how the relative energetic importance of specific amino acid-base pair edge contacts is modulated.
Hilchey
S P
SP
Department of Biological Sciences, State University of New York, Buffalo, New York 14260-1300, USA.
Koudelka
G B
GB
D004247:DNA / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism; D009154:Mutation; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA; D003850:Deoxyribonuclease I