Cell, Vol. 66, 1165-I 195, September
20, 1991, Copyright
0 1991 by Cell Press
A New Mechanism for Coactivation of Transcription Initiation: Repositioning of an Activator Triggered by the Binding of a Second Activator Evelyne Richet, Dominique Vidal-lngigliardi, and Olivier Raibaud Unit6 de Gdnbtique Mol&ulaire URA 1149 du CNRS lnstitut Pasteur 25 rue du Dr. Roux 75724 Paris Cedex 15 France
Summary The CAMP receptor protein (CRP) and MalT, the maltose regulon activator, synergistically activate transcription from the E. coli ma/Kp promoter. The ma/Kp regulatory region comprises two series of MalTbinding sites separated by three CRP-binding sites. By combining genetic and biochemical studies, we demonstrate that the promoter-proximal region contains two overlapping sets of three MaIT-binding sites. Occupation of the higher affinity set of sites, which occurs in the absence of CRP, does not lead to ma/Kp activation. In contrast, in the presence of CRP, MalT binds to the lower affinity set of sites and triggers transcription initiation because, unlikethe highaffinityset, the low affinity set of sites is properly positioned with respect to the Pribnow box. The CRP effect requires the ma/Kpdistal MaIT-binding sites. The synergistic action of MalT and CRP therefore relies on MalT repositioning via the formation of a nucleoprotein structure involving the entire regulatory region.
Introduction Gene expression is often modulated by several transcriptional regulators in response to multiple external signals. The characterization of numerous promoters in prokaryotes has revealed that the way in which the transcription signals are integrated varies greatly from system to system. Nevertheless, one can distinguish two basic strategies. In the first of these, the regulatory proteins work independently either at the same promoter, as observed for the control of the lac promoter by the CAMP receptor protein (CRP) activator and the Lacl repressor, or by specifically controlling different promoters placed in tandem, as observed for the control of the deoCABD operon (ValentinHansen et al., 1982) or that of the g/nA gene (Reitzer and Magasanik, 1985). In the second strategy, the regulatory proteins act synergistically, as observed in the activation of the araBAD promoter by AraC and CRP (Lichenstein et al., 1987; Lobell and Schleif, 1991) or in the corepression of deoP2 by CRP and CytR (S@gaard-Andersen et al., 1991). The regulatory processes involving cooperation between two activators or repressors raise two important questions. What are the underlying mechanisms and how
is specificity achieved? In particular, why is a protein unable to activate (or repress) a given promoter without the help of a second regulatory protein, while it can act alone on another promoter? These problems are particularly relevant in eukaryotes, where gene expression is often under the specific control of different combinations of transcription factors. The maltose regulon in Escherichia coli and Klebsiella pneumoniae constitutes a model system whose study may provide some insight into how a given combination of regulators can specifically activate a subset of genes. The expression of this regulon is controlled by two activator proteins, MalT and CRP. While the activity of some of the ma/ promoters depends only on the presence of MalT (ma//$, pu/Ap, and pu/Cp) or of CRP (ma/Tp), the activity of other promoters depends on the presence of both proteins (ma/Ep and ma/Kp) (Schwartz, 1987). CRP is a pleiotropic regulatory protein that controls carbon source utilization in response to the availability of sugars, such as glucose, that enter the cell via the phosphotransferase system (de Crombrugghe et al., 1984). MalT, the transcriptional activator of the maltose regulon, is a monomeric protein in solution (M, 103,000) and is active only in the presence of ATP and maltotriose, the inducer of the regulon (Raibaud and Richet, 1987; Richet and Raibaud, 1989). MalT recognizes the asymmetrical nucleotide sequence 5’-ggGGAT/ GGAgg, in which the central hexanucleotide (5’-GGAT/ GGA), almost totally conserved in the different MalTbinding sites, seems to play a major role in the interaction with MalT (Vidal-lngigliardi et al., 1991). The malEp and malKp promoters, the activity of which depends on both CRP and MalT, form a pair of divergent promoters that direct the expression of the ma/EFG and malK-IamB-malM operons encoding the maltodextrin transport system. In both cases, MalT is absolutely required for initiation of transcription, a feature common to all of the MalT-controlled promoters, while CRP is an accessory factor. In the absence of CRP, ma/Ep and ma/Kp still work at 20% and 10% efficiency, respectively (Schwartz, 1987; Raibaud et al., 1989; Vidal-lngigliardi and Raibaud, 1991). The inability of CRP alone to activate any of these promoters contrasts with its ability to activate other promoters such as malTp or the lac and gal promoters (Menendez et al., 1987; But et al., 1987). Recent genetic and biochemical studies have shown that the structures of malEp and ma/Kp are rather intricate. Their transcription start sites are 271 bp apart and their activity is controlled by a 210 bp regulatory region located in between and comprising two series of MalT-binding sites separated by three CRPbinding sites (see Figure 1A). The three CRP-binding sites and most, if not all, of the MalT-binding sites are important for the full activity of both promoters (Raibaud et al., 1989; Vidal-lngigliardi and Raibaud, 1991; Vidal-lngigliardi et al., 1991). Most strikingly, ma/Kp activity is reduced to less than 1% by deletion of the ma/Ep-proximal MalT-binding sites, which are located between positions -215 and -240
Cell 1188
A 12
malfp
1
2
3
WYUC
Figure Activity
5/s
a
1. Mutations
Restoring
malKpAl04
malKp (285U) malKpA 104 (
20, 1991, Copyright
0 1991 by Cell Press
A New Mechanism for Coactivation of Transcription Initiation: Repositioning of an Activator Triggered by the Binding of a Second Activator Evelyne Richet, Dominique Vidal-lngigliardi, and Olivier Raibaud Unit6 de Gdnbtique Mol&ulaire URA 1149 du CNRS lnstitut Pasteur 25 rue du Dr. Roux 75724 Paris Cedex 15 France
Summary The CAMP receptor protein (CRP) and MalT, the maltose regulon activator, synergistically activate transcription from the E. coli ma/Kp promoter. The ma/Kp regulatory region comprises two series of MalTbinding sites separated by three CRP-binding sites. By combining genetic and biochemical studies, we demonstrate that the promoter-proximal region contains two overlapping sets of three MaIT-binding sites. Occupation of the higher affinity set of sites, which occurs in the absence of CRP, does not lead to ma/Kp activation. In contrast, in the presence of CRP, MalT binds to the lower affinity set of sites and triggers transcription initiation because, unlikethe highaffinityset, the low affinity set of sites is properly positioned with respect to the Pribnow box. The CRP effect requires the ma/Kpdistal MaIT-binding sites. The synergistic action of MalT and CRP therefore relies on MalT repositioning via the formation of a nucleoprotein structure involving the entire regulatory region.
Introduction Gene expression is often modulated by several transcriptional regulators in response to multiple external signals. The characterization of numerous promoters in prokaryotes has revealed that the way in which the transcription signals are integrated varies greatly from system to system. Nevertheless, one can distinguish two basic strategies. In the first of these, the regulatory proteins work independently either at the same promoter, as observed for the control of the lac promoter by the CAMP receptor protein (CRP) activator and the Lacl repressor, or by specifically controlling different promoters placed in tandem, as observed for the control of the deoCABD operon (ValentinHansen et al., 1982) or that of the g/nA gene (Reitzer and Magasanik, 1985). In the second strategy, the regulatory proteins act synergistically, as observed in the activation of the araBAD promoter by AraC and CRP (Lichenstein et al., 1987; Lobell and Schleif, 1991) or in the corepression of deoP2 by CRP and CytR (S@gaard-Andersen et al., 1991). The regulatory processes involving cooperation between two activators or repressors raise two important questions. What are the underlying mechanisms and how
is specificity achieved? In particular, why is a protein unable to activate (or repress) a given promoter without the help of a second regulatory protein, while it can act alone on another promoter? These problems are particularly relevant in eukaryotes, where gene expression is often under the specific control of different combinations of transcription factors. The maltose regulon in Escherichia coli and Klebsiella pneumoniae constitutes a model system whose study may provide some insight into how a given combination of regulators can specifically activate a subset of genes. The expression of this regulon is controlled by two activator proteins, MalT and CRP. While the activity of some of the ma/ promoters depends only on the presence of MalT (ma//$, pu/Ap, and pu/Cp) or of CRP (ma/Tp), the activity of other promoters depends on the presence of both proteins (ma/Ep and ma/Kp) (Schwartz, 1987). CRP is a pleiotropic regulatory protein that controls carbon source utilization in response to the availability of sugars, such as glucose, that enter the cell via the phosphotransferase system (de Crombrugghe et al., 1984). MalT, the transcriptional activator of the maltose regulon, is a monomeric protein in solution (M, 103,000) and is active only in the presence of ATP and maltotriose, the inducer of the regulon (Raibaud and Richet, 1987; Richet and Raibaud, 1989). MalT recognizes the asymmetrical nucleotide sequence 5’-ggGGAT/ GGAgg, in which the central hexanucleotide (5’-GGAT/ GGA), almost totally conserved in the different MalTbinding sites, seems to play a major role in the interaction with MalT (Vidal-lngigliardi et al., 1991). The malEp and malKp promoters, the activity of which depends on both CRP and MalT, form a pair of divergent promoters that direct the expression of the ma/EFG and malK-IamB-malM operons encoding the maltodextrin transport system. In both cases, MalT is absolutely required for initiation of transcription, a feature common to all of the MalT-controlled promoters, while CRP is an accessory factor. In the absence of CRP, ma/Ep and ma/Kp still work at 20% and 10% efficiency, respectively (Schwartz, 1987; Raibaud et al., 1989; Vidal-lngigliardi and Raibaud, 1991). The inability of CRP alone to activate any of these promoters contrasts with its ability to activate other promoters such as malTp or the lac and gal promoters (Menendez et al., 1987; But et al., 1987). Recent genetic and biochemical studies have shown that the structures of malEp and ma/Kp are rather intricate. Their transcription start sites are 271 bp apart and their activity is controlled by a 210 bp regulatory region located in between and comprising two series of MalT-binding sites separated by three CRPbinding sites (see Figure 1A). The three CRP-binding sites and most, if not all, of the MalT-binding sites are important for the full activity of both promoters (Raibaud et al., 1989; Vidal-lngigliardi and Raibaud, 1991; Vidal-lngigliardi et al., 1991). Most strikingly, ma/Kp activity is reduced to less than 1% by deletion of the ma/Ep-proximal MalT-binding sites, which are located between positions -215 and -240
Cell 1188
A 12
malfp
1
2
3
WYUC
Figure Activity
5/s
a
1. Mutations
Restoring
malKpAl04
malKp (285U) malKpA 104 (
