TY - JOUR
T1 - Dynamic Structural Rearrangements Between DNA Binding Modes of E. coli SSB Protein
AU - Roy, Rahul
AU - Kozlov, Alexander G.
AU - Lohman, Timothy M.
AU - Ha, Taekjip
N1 - Funding Information:
R.R. thanks S. McKinney for data acquisition programs; C. Joo, I. Rasnik, S. Hohng, M. Nahas and S. Myong for experimental help and discussion. We also thank Dr G. Waksman for providing the PDB structures for Figure 1 and T. Ho for DNA synthesis. Funding for the work was provided by NIH grants to T.H. (GM065367) and T.M.L. (GM030498). T.H. is an investigator with the Howard Hughes Medical Institute.
PY - 2007/6/22
Y1 - 2007/6/22
N2 - Escherichia coli single-stranded (ss)DNA binding (SSB) protein binds ssDNA in multiple binding modes and regulates many DNA processes via protein-protein interactions. Here, we present direct evidence for fluctuations between the two major modes of SSB binding, (SSB)35 and (SSB)65 formed on (dT)70, with rates of interconversion on time scales that vary as much as 200-fold for a mere fourfold change in NaCl concentration. Such remarkable electrostatic effects allow only one of the two modes to be significantly populated outside a narrow range of salt concentration, providing a context for precise control of SSB function in cellular processes via SSB expression levels and interactions with other proteins. Deletion of the acidic C terminus of SSB, the site of binding of several proteins involved in DNA metabolism, does not affect the strong salt dependence, but shifts the equilibrium towards the highly cooperative (SSB)35 mode, suggesting that interactions of proteins with the C terminus may regulate the binding mode transition and vice versa. Single molecule analysis further revealed a novel low abundance binding configuration and provides a direct demonstration that the SSB-ssDNA complex is a finely tuned assembly in dynamic equilibrium among several well-defined structural and functional states.
AB - Escherichia coli single-stranded (ss)DNA binding (SSB) protein binds ssDNA in multiple binding modes and regulates many DNA processes via protein-protein interactions. Here, we present direct evidence for fluctuations between the two major modes of SSB binding, (SSB)35 and (SSB)65 formed on (dT)70, with rates of interconversion on time scales that vary as much as 200-fold for a mere fourfold change in NaCl concentration. Such remarkable electrostatic effects allow only one of the two modes to be significantly populated outside a narrow range of salt concentration, providing a context for precise control of SSB function in cellular processes via SSB expression levels and interactions with other proteins. Deletion of the acidic C terminus of SSB, the site of binding of several proteins involved in DNA metabolism, does not affect the strong salt dependence, but shifts the equilibrium towards the highly cooperative (SSB)35 mode, suggesting that interactions of proteins with the C terminus may regulate the binding mode transition and vice versa. Single molecule analysis further revealed a novel low abundance binding configuration and provides a direct demonstration that the SSB-ssDNA complex is a finely tuned assembly in dynamic equilibrium among several well-defined structural and functional states.
KW - DNA-protein interactions
KW - FRET
KW - binding modes
KW - replication
KW - single-stranded DNA binding protein
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U2 - 10.1016/j.jmb.2007.03.079
DO - 10.1016/j.jmb.2007.03.079
M3 - Article
C2 - 17490681
AN - SCOPUS:34248664689
SN - 0022-2836
VL - 369
SP - 1244
EP - 1257
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 5
ER -