COMMENTARY
Increased subtlety of transcription factor binding increases complexity of genome regulation Peter H. von Hippel1 Department of Chemistry and Biochemistry, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403
In PNAS, Afek et al. (1) show that the specific (or “consensus”) base-pair sequences of a regulatory DNA target are not solely responsible for controlling the binding affinities of several eukaryotic transcription factors (TFs) for their DNA target sites. Rather, so-called “nonconsensus” sequences that flank the specific target, particularly flanking sequences that contain certain base-pair repeat symmetries, also seem to play a major role in regulating binding preferences and these effects significantly modulate the measured binding affinities of the TFs tested. Such symmetries also appear to alter the nonspecific binding of TFs to duplex DNA in the absence of specific binding sequences. Why is this important? The chromosomes of higher organisms, as well as the nucleoid bodies of bacteria, can be viewed as giant DNA–protein complexes, with the DNA storing the coding and regulatory information of the cell as defined basepair sequences and the protein components directing the manipulation, expression, and transmission of this information. These giant complexes are also dynamic: changing shape, protein composition, and gene-expression levels as required by cellular metabolic needs and cell-cycle stages. These large-scale cyclical events result in a kaleidoscope of timedependent protein–DNA interactions, and although some stages of these interactions have been captured as “structural snapshots” by X-ray crystallography or cryo-electron microscopy, our understanding of the underlying thermodynamic and kinetic changes that direct these processes is still at a primitive level. Advances in our knowledge of the shifting thermodynamic landscapes that underlie these DNA–protein binding interactions, and the development of new bulk solution and single-molecule methods for studying the kinetics of the transitions between binding states during the events of genome regulation in vivo and in vitro and in real time (for example, see refs. 2–4), may now permit new approaches to these problems.
Shortly after the isolation of the lac and λ transcription repressors and the demonstration that they bind specifically to DNA operators of defined base-pair sequence to regulate access to the promoters of the relevant genes (5, 6), it also became apparent that this specific-site binding was regulated by coupled equilibria involving competitive binding of these TFs to the rest of the duplex DNA (7). Indeed, it turned out that the controlling energetics of the binding of the lac repressor (R) to operator (O) sites, and its removal (induction) from these sites by metabolic intermediates, was tightly coupled to its competitive binding to nonspecific DNA binding sites (8, 9). In addition, early measurements of the on-rate of R binding to its O target appeared to exceed the rate expected for the free 3D diffusion of a protein of that size (10). This finding suggested that the binding pathway must include an intermediate binding target for R—presumably other sequences of the DNA genome—and that final operator location and R–O binding recognition must be facilitated, rather than inhibited, by this transient binding of R to nonspecific sites (11–13). Some structural aspects of the thermodynamic (occupancy) and kinetic (target location and docking) issues involved in these processes are summarized in Fig. 1 A and B, and the thermodynamic issues can be thought of in the context of a plot of fractional saturation (θ) of a specific DNA target site as a function of TF concentration (log [TF]), as shown in Fig. 1C. Clearly, small changes in TF binding affinity can only regulate target site occupancy in a sensitive fashion if the concentration of TF is comparable in magnitude to the dissociation equilibrium constant (Kd,sp). Thus, the TF concentration range spanning the central region of the binding titration curve has been dubbed the “window of specificity” for a given TF (14), and corresponds to the concentration range over which target-
17344–17345 | PNAS | December 9, 2014 | vol. 111 | no. 49
site occupancy can be easily regulated and TF binding can serve as a sensitive regulatory “switch” (6). The binding windows for more weakly bound targets (represented by O2 and O3 in Fig. 1C) occur at higher TF concentrations. Physiological mechanisms that maintain the intracellular concentration of a given TF at values close to its Kd,sp represent one tactic for maintaining the regulatory system within the specificity window. Other mechanisms may involve “moving” the window along the log [TF] axis, making TF binding cooperative, decreasing Kd,sp by mutating the target sequence, or manipulating the nonconsensus contributions to the specific binding affinity of the vicinal DNA sequences (1). Other cellular considerations are involved in setting Kd,sp and establishing the center of the specificity window. The regulated concentrations of many TFs in prokaryote cells or eukaryote nuclei fall in the micromolarto-nanomolar concentration range, which means that if the TF association rate is close to the diffusion-controlled limit, the dissociation rate will be in the millisecond-to-second range, assuring that several cycles of binding and release will occur during the lifetimes of most biological processes, and thus that binding equilibrium (including proper annealing in macromolecular assembly processes) can be achieved. Much tighter specific binding interactions (as in R–O complex formation) require additional processes (induction and nonspecific DNA binding in the case of lac R) to weaken the affinity sufficiently to permit TF release to occur at rates compatible with the cell cycle and other biological time constraints. Assuming that binding and release events do occur rapidly enough for equilibrium to be achieved, additional opportunities to regulate TF DNA site occupancy may involve the competitive binding of proteins or protein complexes (such as nucleosomes manipulated by chromatin Author contributions: P.H.v.H. wrote the paper. The author declares no conflict of interest. See companion article on page 17140 in issue 48 of volume 111. 1
Email: [email protected].
www.pnas.org/cgi/doi/10.1073/pnas.1418978111
1 Afek A, Schipper JL, Horton J, Gordân R, Lukatsky DB (2014) Protein–DNA binding in the absence of specific base-pair recognition. Proc Natl Acad Sci USA 111(48):17140–17145. 2 Hammar P, et al. (2012) The lac repressor displays facilitated diffusion in living cells. Science 336(6088):1595–1598. 3 Bakshi S, Dalrymple RM, Li W, Choi H, Weisshaar JC (2013) Partitioning of RNA polymerase activity in live Escherichia coli from analysis of single-molecule diffusive trajectories. Biophys J 105(12): 2676–2686. 4 Phelps C, Lee W, Jose D, von Hippel PH, Marcus AH (2013) Singlemolecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions. Proc Natl Acad Sci USA 110(43):17320–17325. 5 Gilbert W, Maxam A (1973) The nucleotide sequence of the lac operator. Proc Natl Acad Sci USA 70(12):3581–3584. 6 Ptashne M (2004) A Genetic Switch: Phage Lambda Revisted (Cold Spring Harbor Lab Press, Cold Spring Harbor, NY), 3rd Ed. 7 von Hippel PH, Revzin A, Gross CA, Wang AC (1974) Non-specific DNA binding of genome regulating proteins as a biological control mechanism: I. The lac operon: Equilibrium aspects. Proc Natl Acad Sci USA 71(12):4808–4812. 8 Laiken SL, Gross CA, von Hippel PH (1972) Equilibrium and kinetic studies of Escherichia coli lac repressor-inducer interactions. J Mol Biol 66(1):143–155. 9 Kao-Huang Y, et al. (1977) Nonspecific DNA binding of genomeregulating proteins as a biological control mechanism: measurement
Fig. 1. TF binding interactions with specific and nonspecific DNA. (A) Schematic representations of binding interactions of TF (here represented by Escherichia coli lac R) with various DNA binding sites. Arrows indicate DNA basepair sequence-specific contacts, “+” charge signs indicate basic amino acid residue contacts with DNA phosphates, and recessed symbols indicate interactions that are not “formed” in the complexes shown. Specific TF–DNA binding involves both base-pair–specific and charge–charge contacts, whereas nonspecific binding involves no base-pair– specific contacts and (here, for lac R) additional charge–charge interactions. Intermediate (nonconsensus) binding forms (1) would fall between these extreme forms and likely involve both base-specific and charge–charge interactions. The lower schematic shows a model of the nonspecifically bound form “sliding” on the DNA in a 1D random walk, accompanied by the displacement of condensed monovalent cations from the DNA (12, 18). Reprinted with permission from ref. 12. (B) Known structures for the free, nonspecifically bound, and specifically bound lac R–DNA complexes (19). Reprinted with permission from ref. 19. (C) The “window of specificity” concept in which specific TF target site occupancy is plotted against log [TF] (for details, see text). The top x axis indicates the estimated number of TF molecules present in an idealized E. coli cell (1 μm3 estimated volume) at the TF concentrations shown. These numbers should also apply roughly to a eukaryotic cell nucleus of about this size. Reprinted from ref. 14.
of DNA-bound Escherichia coli lac repressor in vivo. Proc Natl Acad Sci USA 74(10):4228–4232. 10 Riggs AD, Bourgeois S, Cohn M (1970) The lac repressoroperator interaction. 3. Kinetic studies. J Mol Biol 53(3):401–417. 11 Berg OG, Winter RB, von Hippel PH (1981) Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. Biochemistry 20(24):6929–6948. 12 von Hippel PH, Berg OG (1989) Facilitated target location in biological systems. J Biol Chem 264(2):675–678. 13 Halford SE, Marko JF (2004) How do site-specific DNA-binding proteins find their targets? Nucleic Acids Res 32(10):3040–3052. 14 von Hippel PH, Berg OG (1989) DNA-protein interactions in the regulation of gene expression. Protein-Nucleic Acid Interactions, eds Saenger W, Heinemann U (MacMillan, London), pp 1–18. 15 Segal E, Widom J (2009) From DNA sequence to transcriptional behaviour: A quantitative approach. Nat Rev Genet 10(7):443–456. 16 Siggers T, Gordân R (2014) Protein-DNA binding: Complexities
rearrangement factors) to target sites that overlap those of the TFs (15). Variations in nonconsensus binding affinities introduced by flanking symmetry elements, together with structure-based heterogeneities in nonspecific binding as a function of DNA duplex shape and
von Hippel
groove geometry (16, 17), may also modulate the facilitated diffusion processes involved in the translocation of TFs and other regulatory protein complexes during the events of chromatin rearrangement and genome expression. Tighter nonconsensus binding near TF target sites could lengthen
and multi-protein codes. Nucleic Acids Res 42(4):2099–2111. 17 Rohs R, et al. (2010) Origins of specificity in protein-DNA recognition. Annu Rev Biochem 79:233–269. 18 von Hippel PH (2007) From “simple” DNA–protein interactions to the macromolecular machines of gene expression. Annu Rev Biophys Biomol Struct 36:79–105. 19 Kalodimos CG, et al. (2004) Structure and flexibility adaptation in nonspecific and specific protein-DNA complexes. Science 305(5682): 386–389.
PNAS | December 9, 2014 | vol. 111 | no. 49 | 17345
COMMENTARY
the “sliding” pathways of control proteins and facilitate specific TF docking, whereas minor differences in base sequences could alter the rates at which proteins move along the DNA by “sliding” or “hopping” mechanisms. Clearly, effects on the energetics and dynamics of protein–DNA complexes that depend on binding differences of regulatory proteins to nonconsensus DNA sequences (1) will provide many additional opportunities for the evolutionary fine-tuning of genome function and cell-cycle regulation, and add layers of quantitative complexity to efforts to explain these processes.
Increased subtlety of transcription factor binding increases complexity of genome regulation Peter H. von Hippel1 Department of Chemistry and Biochemistry, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403
In PNAS, Afek et al. (1) show that the specific (or “consensus”) base-pair sequences of a regulatory DNA target are not solely responsible for controlling the binding affinities of several eukaryotic transcription factors (TFs) for their DNA target sites. Rather, so-called “nonconsensus” sequences that flank the specific target, particularly flanking sequences that contain certain base-pair repeat symmetries, also seem to play a major role in regulating binding preferences and these effects significantly modulate the measured binding affinities of the TFs tested. Such symmetries also appear to alter the nonspecific binding of TFs to duplex DNA in the absence of specific binding sequences. Why is this important? The chromosomes of higher organisms, as well as the nucleoid bodies of bacteria, can be viewed as giant DNA–protein complexes, with the DNA storing the coding and regulatory information of the cell as defined basepair sequences and the protein components directing the manipulation, expression, and transmission of this information. These giant complexes are also dynamic: changing shape, protein composition, and gene-expression levels as required by cellular metabolic needs and cell-cycle stages. These large-scale cyclical events result in a kaleidoscope of timedependent protein–DNA interactions, and although some stages of these interactions have been captured as “structural snapshots” by X-ray crystallography or cryo-electron microscopy, our understanding of the underlying thermodynamic and kinetic changes that direct these processes is still at a primitive level. Advances in our knowledge of the shifting thermodynamic landscapes that underlie these DNA–protein binding interactions, and the development of new bulk solution and single-molecule methods for studying the kinetics of the transitions between binding states during the events of genome regulation in vivo and in vitro and in real time (for example, see refs. 2–4), may now permit new approaches to these problems.
Shortly after the isolation of the lac and λ transcription repressors and the demonstration that they bind specifically to DNA operators of defined base-pair sequence to regulate access to the promoters of the relevant genes (5, 6), it also became apparent that this specific-site binding was regulated by coupled equilibria involving competitive binding of these TFs to the rest of the duplex DNA (7). Indeed, it turned out that the controlling energetics of the binding of the lac repressor (R) to operator (O) sites, and its removal (induction) from these sites by metabolic intermediates, was tightly coupled to its competitive binding to nonspecific DNA binding sites (8, 9). In addition, early measurements of the on-rate of R binding to its O target appeared to exceed the rate expected for the free 3D diffusion of a protein of that size (10). This finding suggested that the binding pathway must include an intermediate binding target for R—presumably other sequences of the DNA genome—and that final operator location and R–O binding recognition must be facilitated, rather than inhibited, by this transient binding of R to nonspecific sites (11–13). Some structural aspects of the thermodynamic (occupancy) and kinetic (target location and docking) issues involved in these processes are summarized in Fig. 1 A and B, and the thermodynamic issues can be thought of in the context of a plot of fractional saturation (θ) of a specific DNA target site as a function of TF concentration (log [TF]), as shown in Fig. 1C. Clearly, small changes in TF binding affinity can only regulate target site occupancy in a sensitive fashion if the concentration of TF is comparable in magnitude to the dissociation equilibrium constant (Kd,sp). Thus, the TF concentration range spanning the central region of the binding titration curve has been dubbed the “window of specificity” for a given TF (14), and corresponds to the concentration range over which target-
17344–17345 | PNAS | December 9, 2014 | vol. 111 | no. 49
site occupancy can be easily regulated and TF binding can serve as a sensitive regulatory “switch” (6). The binding windows for more weakly bound targets (represented by O2 and O3 in Fig. 1C) occur at higher TF concentrations. Physiological mechanisms that maintain the intracellular concentration of a given TF at values close to its Kd,sp represent one tactic for maintaining the regulatory system within the specificity window. Other mechanisms may involve “moving” the window along the log [TF] axis, making TF binding cooperative, decreasing Kd,sp by mutating the target sequence, or manipulating the nonconsensus contributions to the specific binding affinity of the vicinal DNA sequences (1). Other cellular considerations are involved in setting Kd,sp and establishing the center of the specificity window. The regulated concentrations of many TFs in prokaryote cells or eukaryote nuclei fall in the micromolarto-nanomolar concentration range, which means that if the TF association rate is close to the diffusion-controlled limit, the dissociation rate will be in the millisecond-to-second range, assuring that several cycles of binding and release will occur during the lifetimes of most biological processes, and thus that binding equilibrium (including proper annealing in macromolecular assembly processes) can be achieved. Much tighter specific binding interactions (as in R–O complex formation) require additional processes (induction and nonspecific DNA binding in the case of lac R) to weaken the affinity sufficiently to permit TF release to occur at rates compatible with the cell cycle and other biological time constraints. Assuming that binding and release events do occur rapidly enough for equilibrium to be achieved, additional opportunities to regulate TF DNA site occupancy may involve the competitive binding of proteins or protein complexes (such as nucleosomes manipulated by chromatin Author contributions: P.H.v.H. wrote the paper. The author declares no conflict of interest. See companion article on page 17140 in issue 48 of volume 111. 1
Email: [email protected].
www.pnas.org/cgi/doi/10.1073/pnas.1418978111
1 Afek A, Schipper JL, Horton J, Gordân R, Lukatsky DB (2014) Protein–DNA binding in the absence of specific base-pair recognition. Proc Natl Acad Sci USA 111(48):17140–17145. 2 Hammar P, et al. (2012) The lac repressor displays facilitated diffusion in living cells. Science 336(6088):1595–1598. 3 Bakshi S, Dalrymple RM, Li W, Choi H, Weisshaar JC (2013) Partitioning of RNA polymerase activity in live Escherichia coli from analysis of single-molecule diffusive trajectories. Biophys J 105(12): 2676–2686. 4 Phelps C, Lee W, Jose D, von Hippel PH, Marcus AH (2013) Singlemolecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions. Proc Natl Acad Sci USA 110(43):17320–17325. 5 Gilbert W, Maxam A (1973) The nucleotide sequence of the lac operator. Proc Natl Acad Sci USA 70(12):3581–3584. 6 Ptashne M (2004) A Genetic Switch: Phage Lambda Revisted (Cold Spring Harbor Lab Press, Cold Spring Harbor, NY), 3rd Ed. 7 von Hippel PH, Revzin A, Gross CA, Wang AC (1974) Non-specific DNA binding of genome regulating proteins as a biological control mechanism: I. The lac operon: Equilibrium aspects. Proc Natl Acad Sci USA 71(12):4808–4812. 8 Laiken SL, Gross CA, von Hippel PH (1972) Equilibrium and kinetic studies of Escherichia coli lac repressor-inducer interactions. J Mol Biol 66(1):143–155. 9 Kao-Huang Y, et al. (1977) Nonspecific DNA binding of genomeregulating proteins as a biological control mechanism: measurement
Fig. 1. TF binding interactions with specific and nonspecific DNA. (A) Schematic representations of binding interactions of TF (here represented by Escherichia coli lac R) with various DNA binding sites. Arrows indicate DNA basepair sequence-specific contacts, “+” charge signs indicate basic amino acid residue contacts with DNA phosphates, and recessed symbols indicate interactions that are not “formed” in the complexes shown. Specific TF–DNA binding involves both base-pair–specific and charge–charge contacts, whereas nonspecific binding involves no base-pair– specific contacts and (here, for lac R) additional charge–charge interactions. Intermediate (nonconsensus) binding forms (1) would fall between these extreme forms and likely involve both base-specific and charge–charge interactions. The lower schematic shows a model of the nonspecifically bound form “sliding” on the DNA in a 1D random walk, accompanied by the displacement of condensed monovalent cations from the DNA (12, 18). Reprinted with permission from ref. 12. (B) Known structures for the free, nonspecifically bound, and specifically bound lac R–DNA complexes (19). Reprinted with permission from ref. 19. (C) The “window of specificity” concept in which specific TF target site occupancy is plotted against log [TF] (for details, see text). The top x axis indicates the estimated number of TF molecules present in an idealized E. coli cell (1 μm3 estimated volume) at the TF concentrations shown. These numbers should also apply roughly to a eukaryotic cell nucleus of about this size. Reprinted from ref. 14.
of DNA-bound Escherichia coli lac repressor in vivo. Proc Natl Acad Sci USA 74(10):4228–4232. 10 Riggs AD, Bourgeois S, Cohn M (1970) The lac repressoroperator interaction. 3. Kinetic studies. J Mol Biol 53(3):401–417. 11 Berg OG, Winter RB, von Hippel PH (1981) Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. Biochemistry 20(24):6929–6948. 12 von Hippel PH, Berg OG (1989) Facilitated target location in biological systems. J Biol Chem 264(2):675–678. 13 Halford SE, Marko JF (2004) How do site-specific DNA-binding proteins find their targets? Nucleic Acids Res 32(10):3040–3052. 14 von Hippel PH, Berg OG (1989) DNA-protein interactions in the regulation of gene expression. Protein-Nucleic Acid Interactions, eds Saenger W, Heinemann U (MacMillan, London), pp 1–18. 15 Segal E, Widom J (2009) From DNA sequence to transcriptional behaviour: A quantitative approach. Nat Rev Genet 10(7):443–456. 16 Siggers T, Gordân R (2014) Protein-DNA binding: Complexities
rearrangement factors) to target sites that overlap those of the TFs (15). Variations in nonconsensus binding affinities introduced by flanking symmetry elements, together with structure-based heterogeneities in nonspecific binding as a function of DNA duplex shape and
von Hippel
groove geometry (16, 17), may also modulate the facilitated diffusion processes involved in the translocation of TFs and other regulatory protein complexes during the events of chromatin rearrangement and genome expression. Tighter nonconsensus binding near TF target sites could lengthen
and multi-protein codes. Nucleic Acids Res 42(4):2099–2111. 17 Rohs R, et al. (2010) Origins of specificity in protein-DNA recognition. Annu Rev Biochem 79:233–269. 18 von Hippel PH (2007) From “simple” DNA–protein interactions to the macromolecular machines of gene expression. Annu Rev Biophys Biomol Struct 36:79–105. 19 Kalodimos CG, et al. (2004) Structure and flexibility adaptation in nonspecific and specific protein-DNA complexes. Science 305(5682): 386–389.
PNAS | December 9, 2014 | vol. 111 | no. 49 | 17345
COMMENTARY
the “sliding” pathways of control proteins and facilitate specific TF docking, whereas minor differences in base sequences could alter the rates at which proteins move along the DNA by “sliding” or “hopping” mechanisms. Clearly, effects on the energetics and dynamics of protein–DNA complexes that depend on binding differences of regulatory proteins to nonconsensus DNA sequences (1) will provide many additional opportunities for the evolutionary fine-tuning of genome function and cell-cycle regulation, and add layers of quantitative complexity to efforts to explain these processes.
