A Point Mutation at the C-Terminal Half of the Repressor of Temperate Mycobacteriophage L1 Affects Its Binding to the Operator DNA

Ganguly, Tridib,Chattoraj, Partho,Das, Malabika,Chanda, Palas K.,Mandal, Nitai.C.,Lee, Chia Y.,Sau, Subrata Korean Society for Biochemistry and Molecular Biol 2004 Journal of biochemistry and molecular biology Vol.37 No.6

The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to $42^{\circ}C$. While 40-95% operator-binding activity was shown to be retained at 35 to $42^{\circ}C$ in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to $38^{\circ}C$, although the latter showed only 10% less binding compared to that of the former at $32^{\circ}C$. The CIts391 showed almost no binding at $42^{\circ}C$. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and $42^{\circ}C$. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at $32^{\circ}C$. Interestingly, the repressor-operator complexes preformed at $0^{\circ}C$ have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to $32^{\circ}C$ after preincubation at 42 to $52^{\circ}C$. All these data suggest that the 131st proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end.

A Point Mutation at the C-Terminal Half of the Repressor of Temperate Mycobacteriophage L1 Affects Its Binding to the Operator DNA

( Tridib Ganguly ),( Partho Chattoraj ),( Malabika Das ),( Palas K. Chanda ),( Nitai. C. Mandal ),( Chia Y. Lee ),( Subrata Sau ) 생화학분자생물학회 2004 BMB Reports Vol.37 No.6

The wild-type repressor CI of temperate mycobacteriophage Ll and the temperature-sensitive (ts) repressor CIts391 of a mutant Ll phage, LlcIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to 42C. While 40-95% operator-binding activity was shown to be retained at 35 to 42C in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to 38℃, although the latter showed only 10% less binding compared to that of the former at 32℃. The CIts391 showed almost no binding at 42℃. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and 42℃. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at 32C. Interestingly, the repressor-operator complexes preformed at 0C have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to 32C after preincubation at 42 to 52C. All these data suggest that the 131 proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the LI repressor is located at its N-terminal end.

Cloning and Characterization of the Promoters of Temperate Mycobacteriophage L1

( Chandrani Chattopadhyay ),( Subrata Sau ),( Nitai C. Mandal ) 생화학분자생물학회 2003 BMB Reports Vol.36 No.6

Four putative promoters of the temperate mycobacteriophage L1 were cloned by detecting the β-galactosidase reporter expression in E. coli transformants that carried L1 specific operon-fusion library. All of the four L1 promoters were also found to express differentially in the homologous environment of mycobacteria. Of the four promoters, two were suggested to be the putative early promoters of L1 since they express within 0 to 10 inin of the initiation of the lytic growth of L1. One of the putative early promoters showed a relatively better and almost identical activity in both E. coli and M. smegmatis. By a sequence analysis, we suggest that the L1 insert that contained the stronger early promoter possibly carries two convergent E. coli σ^(70)-like L1 promoters, which are separated from each other by about 300 nucleotides. One of them is the early promoter of L1 as it showed a 100% similarity with the early P_)left) promoter of the homoimmune phage L5. The second promoter, designated P4, was suggested for its appreciable level of reporter activity in the absence of the -10 element of the P_(left) equivalent of L1. By analyzing most of the best characterized mycobacteriophages-specific promoters, including the L1 promoter P4, we suggest that both the - 10 and -35 hexamers of the mycobacteriophage promoters are highly conserved and almost similar to the consensus - 10 and -35 hexamers of the E. coli σ^(70) promoters.

Cloning and Characterization of the Promoters of Temperate Mycobacteriophage L1

Chattopadhyay, Chandrani,Sau, Subrata,Mandal, Nitai C. Korean Society for Biochemistry and Molecular Biol 2003 Journal of biochemistry and molecular biology Vol.36 No.6

Four putative promoters of the temperate mycobacteriophage L1 were cloned by detecting the $\beta$-galactosidase reporter expression in E. coli transformants that carried L1 specific operon-fusion library. All of the four L1 promoters were also found to express differentially in the homologous environment of mycobacteria. Of the four promoters, two were suggested to be the putative early promoters of L1 since they express within 0 to 10 min of the initiation of the lytic growth of L1. One of the putative early promoters showed a relatively better and almost identical activity in both E. coli and M. smegmatis. By a sequence analysis, we suggest that the L1 insert that contained the stronger early promoter possibly carries two convergent E. coli $\sigma^{70}$-like L1 promoters, which are separated from each other by about 300 nucleotides. One of them is the early promoter of L1 as it showed a 100% similarity with the early $P_{left}$ promoter of the homoimmune phage L5. The second promoter, designated P4, was suggested for its appreciable level of reporter activity in the absence of the -10 element of the $P_{left}$ equivalent of L1. By analyzing most of the best characterized mycobacteriophages-specific promoters, including the L1 promoter P4, we suggest that both the -10 and -35 hexamers of the mycobacteriophage promoters are highly conserved and almost similar to the consensus -10 and -35 hexamers of the E. coli $\sigma^{70}$ promoters.

The Bacteriophage λ DNA Replication Protein P Inhibits the oriC DNA-and ATP-binding Functions of the DNA Replication Initiator Protein DnaA of Escherichia coli

( Indrani Datta ),( Subrata Sau ),( Alok Kumar Sil ),( Nitai C. Mandal ) 생화학분자생물학회 2005 BMB Reports Vol.38 No.1

Under the condition of expression of λP protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the λP protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the λ P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of λ P protein with E. call DNA initiator protein DnaA for phage DNA replication has been proposed.