Abstract
Most transcription activator proteins have three important features that can be probed at the molecular level: they bind to specific sequences near promoters, they can be interconverted between active and inactive forms by covalent or noncovalent modification, and, when bound at target promoters, they can stimulate the initiation of transcription by RNA polymerase (1). This chapter is concerned with in vitro methods for measuring the transcription activation function of this important class of proteins. In most cases, these methods are applied to activators that have been substantially purified, and for which the target sequences are known and the binding sites characterized (other chapters in this volume cover methods for locating and investigating the binding sites for such activators). Here we are concerned with the measurement of the products of activation. Because Escherichia coli transcription activators have been studied more than any others, we will take these as the paradigm, though, in principle, the techniques can be applied to any organism for which in vitro systems have been developed.
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References
Raibaud, O. and Schwartz, M. (1984) Positive control of transcription initiation in bacteria. Annu. Rev. Genet. 18, 173–206.
Losick, R. and Chamberlin, M. (eds.) (1976) RNA Polymerase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Zubay, G. (1980) The isolation and properties of CAP, the catabolite gene activator. Methods Enzymol. 65, 856–877.
McClure, W. (1980) Rate-limiting steps in RNA chain initiation. Proc. Natl. Acad. Sci. USA 77, 5634–5638.
Burgess, R. and Jendrisak, J. (1975) A procedure for the rapid, large-scale purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry 14, 4634–4638.
Hager, D., Jun Jin, D., and Burgess, R. (1990) Use of mono Q high resolution ionic exchange chromotography to obtain highly pure and active Escherichia coli RNA polymerase. Biochemistry 29, 7890–7894.
Tang, H., Severinov, K., Goldfarb, A., and Ebright, R. (1995) Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase. Proc. Natl. Acad. Sci. USA 92, 4902–4906.
Fujita, N. and Ishihama, A. (1996) Reconstitution of RNA polymerase. Methods Enzymol. 273, 121–130.
Kolb, A., Kotlarz, D., Kusano, S., and Ishihama, A. (1995) Selectivity of the Escherichia coli RNA polymerase Eσ38 for overlapping promoters and ability to support CRP activation. Nucleic Acids Res. 23, 819–826.
Sambrook, J., Fritsch, E., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Savery, N., Lloyd, G., Kainz, M., Gaal, T., Ross, W., Ebright, R., et al. (1998) Transcription activation at Class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase alpha subunit. EMBO J. 17, 3439–3447.
Herbert, M., Kolb, A., and Buc, H. (1986) Overlapping promoters and their control in Escherichia coli: the gal case. Proc. Natl. Acad Sci. USA 83, 2807–2811.
Hawley, D. and McClure, W. (1982) Mechanism of activation of transcription initiation from theλ PRM promoter. J. Mol. Biol 157, 493–525.
Malan, T., Kolb, A., Buc, H., and McClure. W. (1984) Mechanism of CRP-cAMP activation of lac operon transcription initiation: activation of the P1 promoter. J. Mol. Biol. 180, 881–909.
Leirmo, S. and Gourse, R. (1991) Factor independent activation of E. coli rRNA transcription (I) Kinetic analysis of the role of the upstream activator region and supercoiling on transcription of the rrnB P1 promoter in vitro. J. Mol. Biol. 220, 555–568.
Rhodius, V., West, D., Webster, C., Busby, S., and Savery, N. (1997) Transcription activation at Class II CRP-dependent promoters: the role of different activating regions. Nucleic Acids Res. 25, 326–333.
Leirmo, S., Harrison, S., Cayley, D., and Burgess, R. (1987) Replacement of potassium chloride by potassium glutamate dramatically enhances protein DNA interactions in vitro. Biochemistry 26, 2095–2101.
Hsu, L. (1996) Quantitative parameters for promoter clearance. Methods Enzymol. 273, 59–71.
Chan, B. and Busby, S. (1989) Recognition of nucleotide sequences at the Escherichia coli galactose operon P1 promoter by RNA polymerase. Gene 84, 227–236.
Straney, D. and Crothers, D. (1985) Intermediates in transcription initiation from the E. coli lac UV5 promoter. Cell 43, 449–459.
Krummel, B. and Chamberlin, M. (1992) Structural analysis of ternary complexes of E. coli RNA polymerase: individual complexes halted along different transcription units have distinct and unexpected biochemical properties. J. Mol. Biol. 225, 221–237.
Grimes, E., Busby, S., and Minchin, S. (1991) Different thermal energy requirement for open complex formation by Escherichia coIl RNA polymerase at two related promoters. Nucleic Acids Res. 19, 6113–6118.
Ponnambalam, S. Spassky, A., and Busby, S. (1987) Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters. Interactions with RNA polymerase and the cyclic AMP receptor protein. FEBS Lett. 219, 189–196.
Gaston, K., Bell, A., Kolb, A., Buc, H., and Busby, S. (1990) Stringent spacing requirements for transcription activation by CRP. Cell 62, 733–743.
Chan, B., Minchin, S., and Busby, S. (1990) Unwinding of the duplex DNA during transcription initiation at the Escherichia coli galactose operon overlapping promoters. FEBS Lett. 267, 46–50.
Goodrich, J. and McClure, W. (1992) Regulation of open complex formation at the galactose operon promoters. Simultaneous interaction of RNA polymerase, gal repressor and CAP/cyclic AMP. J. Mol Biol 224, 15–29.
Bertrand-Burggraf, E., Lefevre. J. F., and Daune, M. (1984) A new experimental approach for studying the association between RNA polymerase and the tet promoter of pBR322. Nucleic Acids Res. 12, 1697–1706.
Qi, F., Liu, C., Heath, L., and Turnbough, C. (1996) In vitro assay for reiterative transcription during transcriptional initiation by Escherichia coli RNA polymerase. Methods Enzymol. 273, 71–85.
Menendez, M., Kolb, A., and Buc, H. (1987) A new target for CRP action at the malT promoter. EMBO J. 6, 4227–4234.
Gussin, G. (1996) Kinetic analysis of RNA polymerase-promoter interactions. Methods in Enzymology 273, 45–59.
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© 2001 Humana Press Inc., Totowa, NJ
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Rhodius, V., Savery, N., Kolb, A., Busby, S. (2001). Assays for Transcription Factor Activity. In: Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology, vol 148. Humana Press. https://doi.org/10.1385/1-59259-208-2:451
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DOI: https://doi.org/10.1385/1-59259-208-2:451
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