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Transcription Activation by Catabolite Activator Protein (CAP)

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Transcription Activation by Catabolite Activator Protein (CAP) ArticleNo.jmbi.1999.3161availableonlineathttp://www.idealibrary.comon J. Mol. Biol. (1999) 293, 199±213 Transcription Activation by Catabolite Activator Protein (CAP) SteveBusby1andRichardH.Ebright2* 1School of Biosciences, The Transcription activation by Escherichia coli catabolite activator protein University of Birmingham (CAP) at each of two classes of simple CAP-dependent promoters is Birmingham B15 2TT, UK understood in structural and mechanistic detail. At class I CAP-depen- dent promoters, CAP activates transcription from a DNA site located 2Howard Hughes Medical upstream of the DNA site for RNA polymerase holoenzyme (RNAP); at Institute, Waksman Institute these promoters, transcription activation involves protein-protein inter- and, Department of Chemistry actions between CAP and the RNAP a subunit C-terminal domain that Rutgers University, New facilitate binding of RNAP to promoter DNA to form the RNAP-promo- Brunswick, NJ 08855, USA ter closed complex. At class II CAP-dependent promoters, CAP activates transcription from a DNA site that overlaps the DNA site for RNAP; at these promoters, transcription activation involves both: (i) protein-protein interactions between CAP and RNAP a subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promo- ter closed complex; and (ii) protein-protein interactions between CAP and RNAP a subunit N-terminal domain that facilitates isomerization of the RNAP-promoter closed complex to the RNAP-promoter open com- plex. Straightforward combination of the mechanisms for transcription activation at class I and class II CAP-dependent promoters permits syner- gistic transcription activation by multiple molecules of CAP, or by CAP and other activators. Interference with determinants of CAP or RNAP involved in transcription activation at class I and class II CAP-dependent promoters permits ``anti-activation'' by negative regulators. Basic features of transcription activation at class I and class II CAP-dependent promo- ters appear to be generalizable to other activators. # 1999 Academic Press Keywords: catabolite activator protein (CAP); cyclic AMP receptor protein *Corresponding author (CRP); RNA polymerase; transcription initiation; transcription activation Introduction (reviewedbyKolbetal.,1993a;Ebright,1993; Busby&Ebright,1997). CAP has provided a classic model system for The Escherichia coli catabolite activator protein structural and mechanistic studies of transcription (CAP; also known as the cAMP receptor protein, activation. Thus, CAP was the ®rst transcription CRP) activates transcription at more than a 100 activatortohavebeenpuri®ed(Zubayetal.1970; promoters. CAP functions by binding, in the pre- Emmeretal.,1970)andthe®rsttranscriptionacti- sence of the allosteric effector cAMP, to speci®c vator to have its three-dimensional structure deter- DNA sites in or near target promoters and enhan- mined(McKay&Steitz,1981),andtranscription cing the ability of RNA polymerase holoenzyme activation by CAP has been the subject of extensive (RNAP) to bind and initiate transcription biophysical, biochemical, and genetic investi- gations(Kolbetal.,1993a;Ebright,1993;Busby& Ebright,1997). Abbreviations used: CAP, catabolite activator protein; CRP, cAMP receptor protein; RNAP, RNA polymerase; Transcription activation by CAP at the sim- aNTD, a subunit N-terminal domain; aCTD, a subunit plest CAP-dependent promoters requires only C-terminal domain; AR1, activating region 1. three macromolecular components (CAP, RNAP, E-mail address of the corresponding author: and promoter DNA) and requires only one DNA [email protected] siteforCAP(Ebright,1993;Busby&Ebright, 0022-2836/99/420199±15 $30.00/0 # 1999 Academic Press 200 Transcription Activation by CAP 1997).TranscriptionactivationbyCAPatsuch promoters is simpler than most examples of transcription activation in bacteria (which require more numerous macromolecular components and/orDNAsites;Gralla&Collado-Vides, 1996),andverysubstantiallysimplerthan examples of transcription activation in eukar- yotes (which require dozens of macromolecular componentsandDNAsites;Roeder,1996; Orphanidesetal.,1997).Accordingly,ithasbeen possible to develop structural and mechanistic descriptions of transcription activation by CAP that are more nearly complete than descriptions of any other examples of transcription activation. In this review, we introduce the three macromol- ecular components required for transcription acti- vation at the simplest CAP-dependent promoters (CAP, RNAP, and promoter DNA), and we present structural and mechanistic descriptions of tran- scription activation at each of two classes of simple CAP-dependent promoters. In addition, we show that basic principles derived from study of tran- scription activation at simple CAP-dependent pro- moters can illuminate understanding of transcription activation at more complex CAP- dependent promoters and at other activator-depen- dent promoters. Macromolecular components CAP CAP has a molecular mass of 45 kDa and is a dimeroftwoidenticalsubunits(Kolbetal.,1993a). Each subunit consists of two domains. The N-term- Figure 1. Structure of the CAP-DNA complex show- inal domain (residues 1-139) is responsible for ing determinants of CAP involved in transcription dimerization of CAP and for interaction with the activation (CAP, light blue; DNA and cAMP bound to CAP,red;Schultzetal.,1991;Parkinsonetal.,1996a).(a) allosteric effector cAMP (which binds to CAP and Determinant of CAP for transcription activation at class induces a conformational change, resulting in a I CAP-dependent promoters (AR1 of downstream conformation competent for DNA binding). The C- subunit, dark blue). (b) Determinants of CAP for tran- terminal domain (residues 140-209) is responsible scription activation at class II CAP-dependent promoters for interaction with DNA, mediating interaction (AR1 of upstream subunit, dark blue; AR2 of down- with DNA through a helix-turn-helix DNA-binding stream subunit, green; AR3 of downstream subunit, motif (for reviews of the helix-turn-helix motif, see yellow). Brennan,1991,1992).CAPrecognizesa22bp, 2-fold-symmetric DNA site (consensus sequence 50- AAATGTGATCTAGATCACATTT-30). The crystallographic structure of CAP has been determined(McKay&Steitz,1981),andseveral RNAP crystallographic structures of CAP in complex with DNAhavebeendetermined(Figure1;Schultzetal., RNAP has a molecular mass of 450 kDa and has 0 1991;Parkinsonetal.1996a,b;Passner&Steitz, subunit composition a2bb s(Chamberlin,1976; 1997;S.Chen,G.Parkinson,J.Liu,B.Benoff,H. Burgess,1976). Berman & R.H.E., unpublished results). The CAP- The a subunit (37 kDa) is responsible for recog- DNA complex is 2-fold symmetric: one CAP sub- nition of the UP element (a supplementary promo- unit interacts with one half of the DNA site, and ter element located upstream of the 35 element in the other CAP subunit interacts in a 2-fold sym- certainpromoters;Rossetal.,1993),andfor metry-related fashion with the other half of the response to a large subset of activators, repressors, DNA site. CAP sharply bends DNA in the CAP- elongationfactors,andterminationfactors(Busby DNA complex, bending DNA to an angle of 80 . &Ebright,1994;Ebright&Busby,1995; The orientation of the CAP-induced DNA bend is Hochschild&Dove,1998;Liu&Hanna,1995;Liu such that the DNA wraps toward and around the etal.,1996;Schaueretal.,1996;Kainz&Gourse, sides of CAP. 1998).Theasubunitconsistsoftwoindependently Transcription Activation by CAP 201 foldeddomains(Blatteretal.,1994;Negishietal., ition 72, or position 62. The best-characterized 1995;Busby&Ebright,1994;Ebright&Busby, class I CAP-dependent promoters are the lac pro- 1995).TheasubunitN-terminaldomain(aNTD; moterandthearti®cialpromoterCC(61.5) residues 8-235) contains the primary determinant (Gastonetal.,1990),eachofwhichhasaDNAsite for dimerization of a, the primary determinant for for CAP centered at position 61.5. interaction of a with the remainder of RNAP, and (ii) Class II CAP-dependent promoters require a determinant for interaction with activators. The a only CAP for transcription activation, and have a subunit C-terminal domain (aCTD; residues 249- single DNA site for CAP overlapping the DNA site 329) contains a secondary, weak determinant for for RNAP, apparently replacing the promoter 35 dimerization of a and determinants for interactions element. The best-characterized class II CAP- with DNA (including sequence-speci®c interactions dependent promoters are the galP1 promoter and with UP-element DNA and non-speci®c inter- thearti®cialpromoterCC(41.5)(Gastonetal., actions with non-UP-element DNA), activators, 1990),eachofwhichhasaDNAsiteforCAPcen- repressors, elongation factors, and termination fac- tered at position 41.5. tors. The linker between aNTD and aCTD is at (iii) Class III CAP-dependent promoters require least 13 amino acid residues in length (544 AÊ if multiple activator molecules for full transcription fully extended) and is unstructured and ¯exible activation, i.e. two or more CAP molecules, or one (Blatteretal.,1994;Negishietal.,1995;Jeonetal., or more CAP molecule and one or more regulon- 1997).Thelong,unstructured,¯exiblelinkerallows speci®c activator molecule. Examples include the aCTD to occupy different positions relative to ansBpromoter(Scottetal.,1995),thearaBADpro- aNTD, and thus relative to the remainder of moter(Lobell&Schleif,1991;Zhang&Schleif, RNAP,indifferenttranscriptioncomplexes(Blatter 1998),themalKpromoter(Richetetal.,1991),and etal.,1994;Busby&Ebright,1994;Ebright&
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