Effects of physical, ionic, and structural factors on the binding of repressor of mycobacteriophage L1 to its cognate operator DNA.
To determine the factors influencing the binding of L1 repressor to its cognate operator DNA, several gel shift as well as bioinformatic analyses have been carried out. The data show that time, temperature, salt, and pH each greatly affect the binding. In order to achieve optimum operator binding of L1 repressor in Tris buffer, the minimum requirements of time, temperature, salt, and pH were estimated to be 1 min, 32 degrees C, NaCl (50 mM), and 7.9, respectively. Interestingly Na+ but not NH4+, K+, or Li+ was found to augment significantly the binding activity of CI protein above the basal level. Anions like Cl-, citrate-, acetate-, and H2PO4- do not alter the binding of L1 repressor to its operator. We also show that an in frame deletion mutant of L1 repressor which does not carry the putative HTH motif (at its N-terminal end) fails to bind to its cognate operator DNA even at very high concentrations. The putative HTH motif was found highly conserved and evolutionarily very close to that of regulatory proteins of Y. pestis, H. marismortui, A. tumefaciens, etc. Taken together we suggest that N-terminal end of L1 repressor carries a HTH motif. Further analysis of the putative secondary structures of mycobacteriophage repressors reveals that two common regions encompassing more than 90% of primary sequence are present in all the four repressor molecules studied here. The results suggest that these common regions are utilized for carrying out identical functions.
Ganguly
Tridib
T
Department of Biochemistry, Bose Institute, P1/12 - CIT Scheme VII M, Calcutta 700 054, India.
Chanda
Palas K
PK
Bandhu
Amitava
A
Chattoraj
Partho
P
Das
Malabika
M
Sau
Subrata
S
D000595:Amino Acid Sequence; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D019077:Helix-Turn-Helix Motifs / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009160:Mycobacteriophages / Q000235:genetics / Q000378:metabolism*; D009875:Operator Regions, Genetic / Q000235:genetics; D010802:Phylogeny; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012492:Salts / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D004279:DNA, Viral; D012097:Repressor Proteins; D012492:Salts; D012965:Sodium Chloride