The effect of nonspecific binding of lambda repressor on DNA looping dynamics.
The λ repressor (CI) protein-induced DNA loop maintains stable lysogeny, yet allows efficient switching to lysis. Herein, the kinetics of loop formation and breakdown has been characterized at various concentrations of protein using tethered particle microscopy and a novel, to our knowledge, method of analysis. Our results show that a broad distribution of rate constants and complex kinetics underlie loop formation and breakdown. In addition, comparison of the kinetics of looping in wild-type DNA and DNA with mutated o3 operators showed that these sites may trigger nucleation of nonspecific binding at the closure of the loop. The average activation energy calculated from the rate constant distribution is consistent with a model in which nonspecific binding of CI between the operators shortens their effective separation, thereby lowering the energy barrier for loop formation and broadening the rate constant distribution for looping. Similarly, nonspecific binding affects the kinetics of loop breakdown by increasing the number of loop-securing protein interactions, and broadens the rate constant distribution for this reaction. Therefore, simultaneous increase of the rate constant for loop formation and reduction of that for loop breakdown stabilizes lysogeny. Given these simultaneous changes, the frequency of transitions between the looped and the unlooped state remains nearly constant. Although the loop becomes more stable thermodynamically with increasing CI concentration, it still opens periodically, conferring sensitivity to environmental changes, which may require switching to lytic conditions.
Physics Department, Emory University, Atlanta, Georgia, USA.
Carlo
C
Manzo
Chiara
C
Zurla
David D
DD
Dunlap
Laura
L
Finzi
D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism*
D004269:DNA, Bacterial; D012097:Repressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins