Regions and residues of an asymmetric operator DNA interacting with the monomeric repressor of temperate mycobacteriophage L1.
Previously, the repressor protein of mycobacteriophage L1 bound to two operator DNAs with dissimilar affinity. Surprisingly, the putative operator consensus sequence, 5'GGTGGa/cTGTCAAG, lacks the dyad symmetry reported for the repressor binding operators of lambda and related phages. To gain insight into the structure of the L1 repressor-asymmetric operator DNA complex, we have performed various in vitro experiments. A dimethyl sulfate protection assay revealed that five guanine bases, mostly distributed in the two adjacent major grooves of the 13 bp operator DNA helix, participate in repressor binding. Hydroxyl radical footprinting demonstrated that interaction between the repressor and operator DNA is asymmetric in nature and occurs primarily through one face of the DNA helix. Genetic studies not only confirmed the results of the dimethyl sulfate protection assay but also indicated that other bases in the 13 bp operator DNA are critical for repressor binding. Interestingly, repressor that weakly induced bending in the asymmetric operator DNA interacted with this operator as a monomer. The tertiary structure of the L1 repressor-operator DNA complex therefore appears to be distinct from those of the lambdoid phages even though the number of repressor molecules per operator site closely matched that of the lambda phage system.
Bandhu
Amitava
A
Department of Biochemistry, Bose Institute, P1/12-CIT Scheme VII M, Kolkata, WB 700 054, India.
Ganguly
Tridib
T
Jana
Biswanath
B
Mondal
Rajkrishna
R
Sau
Subrata
S
D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009160:Mycobacteriophages / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D012097:Repressor Proteins; D014764:Viral Proteins; D004247:DNA