Transcription Activation by Catabolite Activator Protein (CAP)

ArticleNo.jmbi.1999.3161availableonlineathttp://www.idealibrary.comon J. Mol. Biol. (1999) 293, 199±213

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-dependent 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-promoter 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 complex. Straightforward combination of the mechanisms for transcription activation at class I and class II CAP-dependent promoters permits synergistic 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 promoters 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 macromolecular components required for transcription activation at the simplest CAP-dependent promoters (CAP, RNAP, and promoter DNA), and we present structural and mechanistic descriptions of transcription activation at each of two classes of simple CAP-dependent promoters. In addition, we show that basic principles derived from study of transcription activation at simple CAP-dependent promoters can illuminate understanding of transcription activation at more complex CAP- dependent promoters and at other activator-dependent 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& theuhpTpromoter(Merkeletal.,1995). Busby,1995). The b (151 kDa) and b0 (155 kDa) subunits are Transcription activation at class I responsible for the catalytic activity of RNAP and CAP-dependent promoters for response to a subset of activators, repressors, elongation factors, and termination factors CAP determinants (Chamberlin,1976;Severinovetal.,1994;Miller etal.,1997;Nechaev&Severinov,1999). Transcription activation at the lac promoter The s70 subunit (70 kDa) (in this review, we requires a determinant consisting of residues 156- refer only to s70, the principal s subunit) is respon- 164 of CAP, located within the C-terminal domain sible for recognition of the promoter 35 element of CAP, immediately preceding the helix-turn-helix and 10 element (recognised by s70 region 4 and DNA-binding motif of CAP (``activating region 1``, s70 region 2, respectively) and for response to a AR1;Belletal.,1990;Eschenlauer&Reznikoff, subsetofactivators(Busby&Ebright,1994;Gross 1991;Zhouetal.,1993a;Niuetal.,1994).Single etal.,1998). amino acid substitutions within AR1 reduce or Low-resolution structures of RNAP and of eliminate transcription activation at the lac promo- 0 RNAP core (subunit composition a2bb ) have been ter, but does not affect DNA binding and DNA determined using electron microscopy and image bendingbyCAP(Zhouetal.,1993a).Alaninescan- reconstruction(Polyakovetal.,1995;Darstetal., ning indicates that the side-chain of Thr158 is the 1998).Recently,ahigh-resolutionstructureof most important side-chain for function of AR1 RNAP core has been determined using X-ray crys- (Niuetal.,1994).AR1foldsasacanonicaltypeIb- tallography (S. Darst, personal communication). In turn and forms a prominently exposed surface addition, high-resolution structures of aNTD patch with dimensions of 11 AÊ Â14 AÊ (Zhang&Darst,1998),aCTD(Jeonetal.,1995;J. (Figure1(a)).Experimentswith``orientedheterodi- Liu, G. Parkinson, E. Blatter, H. Berman, & R.H.E., mers'' of CAP having one subunit with a func- unpublishedresults),ands70region2(Malhotra tional AR1 and one subunit with a non-functional etal.,1996)havebeendetermined. AR1 indicate that transcription activation at lac requires a functional AR1 only in the downstream CAP-dependent promoters subunitoftheCAPdimer(Figure1(a);Zhouetal., 1993b). CAP-dependent promoters can be grouped into AR1 is essential for transcription activation, not threeclasses(Ushida&Aiba,1990;Ebright,1993): only at lac and other class I CAP-dependent pro- (i) class I CAP-dependent promoters require only moters in which the DNA site for CAP is centered CAP for transcription activation, and have a single near position 62, but also at class I CAP-depen- DNA site for CAP located upstream of the DNA dent promoters in which the DNA site for CAP is site for RNAP. The DNA site for CAP can be centeredfurtherupstream(e.g.nearposition93, located at various distances from the transcription position83,orposition72)(Zhouetal.,1994a). start point, provided that the DNA site for CAP Oriented-heterodimer analysis indicates that, in and the DNA site for RNAP are on the same face each case, AR1 is functionally presented in the of the DNA helix. Thus, the DNA site for CAP can downstreamsubunitoftheCAPdimer(Zhouetal., be centered near position 93, position 83, pos- 1994b). 202 Transcription Activation by CAP

RNAP determinants (ii) Residues in the ``261 determinant'' (Val257, Asp258, Asp259, Glu261) form a surface with dimensions 7AÊ Â16 AÊ that is adjacent to, but Transcription activation at the lac promoter distinct from, the 265 determinant. The 261 deter- requiresaCTD(Igarashi&Ishihama,1991).Thus, minant is not required for aCTD-DNA interaction RNAP reconstituted with truncated a subunits (Tangetal.,1994),but,nevertheless,isimportant lacking CTD is defective in CAP-dependent tran- for UP-element-dependent transcription as well as scription at lac, but not defective in CAP-indepen- CAP-dependent transcription (W. Ross & R. dent transcription at lacUV5 (a CAP-independent Gourse, unpublished; H. Chen & R.H.E., unpub- mutantoflac;Beckwithetal.,1972).Experiments lished). with ``oriented-alpha'' RNAP derivatives having (iii) Residues in the ``287 determinant'' (Thr285, one full-length a subunit and one truncated a sub- Glu286, Val287, Glu288, Leu289, Gly315, Arg317, unit lacking CTD indicate that only one of the two Leu318) form a surface with dimensions copies of aCTD in RNAP is essential for transcrip- 11 AÊ Â22 AÊ that is adjacent to, but distinct tion activation at lac, and that this copy can be, I from, the 265 determinant, and is on the face of interchangeably, aCTD (aCTD of the a subunit aCTD opposite the 261 determinant. The 287 deter- that interacts with b)oraCTDII (aCTD of the a 0 minant is not required for aCTD-DNA interaction subunitthatinteractswithb )(Zouetal.,1993;W. and plays no role in UP-element-dependent tran- Niu & R.H.E., unpublished). scription(Gaaletal.,1996;Saveryetal.,1998).Sub- Isolation and characterization of single amino stitutions in the 287 determinant reduce or acid substitutions in aCTD that result in speci®c eliminate CAP-a cooperativity in experiments asses- defects in class I CAP-dependent transcription at singformationofCAP-a-DNAcomplexes(Savery lac has led to the identi®cation of three critical etal.,1998).Therefore,itisproposedthatthe287 determinants within aCTD, each named by the determinant is essential for protein-protein inter- position at which substitutions result in the most actions between CAP and a on promoter DNA. severedefects(Figure2;Zouetal.,1992;Tangetal., 1994;Murakamietal.,1996;Saveryetal.,1998;N. Savery, R. Gourse, R.H.E. & S.B., unpublished results): DNA determinants (i) Residues in the ``265 determinant'' (Arg265, Hydroxyl radical and DNase I DNA footprinting Asn268, Asn294, Gly296, Lys298, Ser299, Glu302) Ê Ê experiments indicate that formation of the ternary form a surface with dimensions 14 A Â23 A. complex of CAP, RNAP, and the lac promoter The 265 determinant is required for aCTD-DNA results in protection, not only of the DNA site for interaction(Gaaletal.,1996;Murakamietal.,1996). CAP and the core promoter, but also of the DNA Accordingly,the265determinantisinvolvedin segment immediately downstream of the DNA site UP-element-dependent transcription as well as in forCAP(positions50to41;Kolbetal.,1993b). CAP-dependenttranscription(Gaaletal.,1996; Full protection of this DNA segment requires the Murakamietal.,1996). determinants in CAP and RNAP described above; thus, substitution of AR1 results in a reduction of protection, and removal of aCTD results in the completelossofprotection(Kolbetal.,1993b). Transcription activation at the lac promoter is sensitive to the structural integrity of the DNA segment immediately downstream of the DNA site for CAP (the same DNA segment protected in the DNA footprinting experiments). Thus, gaps in this DNA segment reduce or eliminate transcription activation(Ryuetal.,1994).Transcriptionacti- vation at the lac promoter also is sensitive to the sequence of this DNA segment. In the wild-type lac promoter, this DNA segment does not corre- spond to a high-af®nity DNA site for aCTD and, indeed, appears to contain no speci®c sequence information(Flatowetal.,1996;Czarnieckietal., 1997).However,replacementofthisDNAsegment by a high-af®nity DNA site for aCTD, i.e. an UP-element subsite (consensus sequence, 50- AAAAAARNA-30;Estremetal.,1999)resultsinan Figure 2. Structure of aCTD showing determinants increaseintranscription(Czarnieckietal.,1997; involved in CAP-dependent transcription (265 determi- Noel&Reznikoff,1998;seealsoSaveryetal.,1995; nant, red; 261 determinant, blue; 287 determinant, yel- Lawetal.,1999).Theoptimalspacingbetweenthe low;Jeonetal.,1995;J.Liu,G.Parkinson,E.Blatter,H. DNA site for CAP and the UP-element subsite Berman & R.H.E., unpublished results). appearstobefourto®vebase-pairs(Czarniecki Transcription Activation by CAP 203 etal.,1997;seealsoSaveryetal.,1995;Lawetal., 72), CAP is proposed to activate transcription by 1999). making a protein-protein interaction between AR1 of the downstream subunit of the CAP dimer and Mechanism aCTD analogous to that at the lac promoter, caus- ing aCTD to make a protein-DNA interaction with Protein-protein photocrosslinking indicates that, the DNA segment immediately downstream of intheternarycomplexofCAP,RNAP,andthelac CAP analogous to that at the lac promoter promoter, AR1 of CAP is in direct physical proxi- (Figure3(b);Zhouetal.,1994a,b;Blatteretal., mitytoaCTDofRNAP(Chenetal.,1994).Further- 1994).Theunstructured,¯exiblelinkerbetweenthe more, protein-DNA photocrosslinking indicates aCTDandtheremainderofRNAP(Blatteretal., that, in the complex, aCTD is in direct physical 1994;Negishietal.,1995;Jeonetal.,1997),possibly proximity to the DNA segment immediately down- together with bending of the intervening DNA, is stream of the DNA site for CAP (N. Naryshkin, A. proposed to permit establishment at such promo- Revyakin, Y. Kim & R.H.E., unpublished results). ters of the same local CAP-aCTD and aCTD-DNA Together with the above-described results de®ning interactions as at the lac promoter, despite the critical determinants of CAP, RNAP, and promoter difference in position of the DNA site for CAP (cf. DNA, the photocrosslinking results lead to the pro- Figures3(a)and(b)).Atsuchpromoters,asatthe posal that transcription activation at the lac promo- lac promoter, CAP-aCTD interaction is proposed to ter involves a direct protein-protein interaction increase the af®nity of RNAP for promoter DNA, between AR1 of the downstream subunit of the resulting in an increase in KB and, thus, an increase CAP dimer and one of the two copies of aCTD of in transcription. (In the special case of the malT RNAP (interchangeably aCTDI or aCTDII) that promoter, which has a DNA site for CAP centered facilitates binding of that copy of aCTD to the at position 70.5 and an inhibitory high-af®nity DNA segment immediately downstream of CAP DNA site for aCTD centered near position 47, (i.e. the DNA segment between the downstream CAP-aCTD interaction serves to prevent aCTD subunit of the CAP dimer and s70 region 4 bound from interacting with the inhibitory high-af®nity atthepromoter35element)(Figure3(a)). DNA site, and thereby to prevent formation of a The 287 determinant of aCTD is proposed to non-productive RNAP-promoter complex de®cient mediate the protein-protein interaction with AR1 inpromoterescape(Tagami&Aiba,1998,1999).) of CAP, and the 265 determinant of aCTD is pro- At class I CAP-dependent promoters, all CAP- posed to mediate the protein-DNA interaction with RNAP and RNAP-DNA interactions essential for the DNA segment immediately downstream of transcription activation are made downstream of CAP(Figure3(a)).Theroleofthe261determinant the DNA site for CAP, and thus downstream of of aCTD is not completely understood. Preliminary thelocusofCAP-inducedDNAbending(Figure3; results suggest that the 261 determinant mediates a Zhouetal..1993b,1994b;W.Niu,N.Naryshkin, protein-protein interaction with s70 region 4 bound A. Revyakin, Y. Kim & R.H.E., unpublished atthepromoter35element(Figure3(a);W.Ross results). Therefore, it appears unlikely that CAP- &R.Gourse,unpublishedresults;H.Chen,H. induced DNA bending plays an essential role in Tang & R.H.E., unpublished results). transcription activation at class I CAP-dependent The interactions between CAP and RNAP at the promoters. Consistent with this inference, mutants lac promoter increase the af®nity of RNAP for pro- of CAP that result in a decrease in CAP-induced moter DNA, resulting in an increase in the binding DNA bending are not defective in transcription constant, KB, for formation of the RNAP-promoter activation at lac, neither in vivo nor in vitro (A. closed complex and, thus, an increase in transcrip- Kapanidis & R.H.E., unpublished results). tioninitiation(Malanetal.,1984;seealsoRenetal., 1988;Straneyetal.,1989;Kolbetal.,1993b; Transcription activation at class II CAP- Heyduketal.,1993).Allavailabledataareconsist- dependent promoters ent with the proposal that CAP activates transcription at lac solely by helping ``recruit'' RNAP to CAP determinants promoter DNA. In particular, removal of CAP from the pre-formed CAP-RNAP-promoter open Transcription activation at class II CAP-depen- complex (by addition of high concentrations of dent promoters requires two distinct determinants heparin) has no negative effects on subsequent inCAP(Figure1(b)): steps in transcription initiation, elongation, and ter- (i) Transcription activation at class II CAP-depen- mination(Tagami&Aiba,1995). dent promoters, like transcription activation at class At other class I CAP-dependent promoters in ICAP-dependentpromoters,requiresAR1(Bell which the DNA site for CAP is located near pos- etal.,1990;Williamsetal.,1991;Westetal.,1993; ition 62, the mechanism of transcription acti- Zhouetal.,1994a).Oriented-heterodimeranalysis vation is proposed to be identical with that at the indicates that, at class II CAP-dependent promoters, lacpromoter(Zhouetal.,1994a,b). AR1 is functionally presented by the upstream sub- At class I CAP-dependent promoters in which unitoftheCAPdimer(Zhouetal.,1994b). the DNA site for CAP is located further upstream (ii) Transcription activation at class II CAP- (e.g. near position 93, position 83, or position dependent promoters requires a class II-speci®c 204 Transcription Activation by CAP

Figure 3. Transcription activation at class I CAP-dependent promoters. aCTD, aNTD, b, b0, and s denote, respectively, the RNAP a subunit C-terminal domain, the RNAP a subunit N-terminal domain, and the RNAP b, b0, and s70 subunits. aCTD is an indepen- dently folded module and is con- nected to aNTD, and thus to the remainder of RNAP, through a unstructured, ¯exible linker; alternative positioning of aCTD is facilitated by the linker and by bending of the intervening DNA (Blatteretal.,1994;Busby& Ebright,1994;Ebright&Busby, 1995).Forsimplicity,DNAis drawnstraight;inpointoffact,bothCAPandRNAPbendDNA(Schultzetal.,1991;Parkinsonetal.,1996a,b;Rees etal.,1993;Meyer-Almesetal.,1994;Rivettietal.,1999).(a)TernarycomplexofCAP,RNAP,andaclassICAP- dependent promoter having the DNA site for CAP centered near position 62, e.g. lac, CC(61.5). Transcription activation involves direct protein-protein interaction between AR1 of the downstream subunit of CAP (open, dashed circle) and the 287 determinant of one copy of aCTD. The AR1-aCTD interaction facilitates binding of aCTD, through its 265 determinant, to the DNA segment immediately downstream of CAP and, possibly, through its 261 determinant, to s70 region 4 bound at the 35 element. The location of the second, non-contacted copy of aCTD has not been determined de®nitively; therefore, the second copy of aCTD is drawn arbitrarily behind the ®rst. Available evidence suggests that the second copy of aCTD may interact, through its 265 determinant, with the DNA segment upstreamoftheDNAsiteforCAP(Kolbetal.,1993b;N.N.,A.R.,Y.K.&R.H.E.,unpublishedresults).(b)Ternary complex of CAP, RNAP, and a class I CAP-dependent promoter having the DNA site for CAP centered near position 103, position 93, position 83, or position 72, e.g. malT, CC(71.5). Transcription activation involves the same AR1-aCTD and aCTD-DNA interactions as in (a). Available evidence suggests that the second, non-contacted copy of aCTD may interact, through its 265 determinant, with the DNA segment immediately upstream of the 35 element and,possibly,throughits261determinant,withs70region4boundatthe35element(Eichenbergeretal.,1996; Lawetal.,1999;N.S.&S.B.,unpublishedresults).(AdaptedfromBlatteretal.,1994;Zhouetal.,1994a,b;additions basedonSaveryetal.,1998;N.Savery,W.Ross,R.Gourse,R.H.E.&S.B.,unpublishedresults;andH.C.,H.T.& R.H.E., unpublished results.) determinant consisting of residues His19, His21, positive charge is critical for AR2 function. Glu21, and Lys101 of CAP, located in the N-term- Oriented-heterodimer analysis indicates that AR2 inal, cAMP-binding domain of CAP (``activating is functionally presented by the downstream sub- region2``,AR2;Niuetal.,1996).AR2wasident- unitoftheCAPdimer(Williamsetal.,1996;Niu i®ed by isolation of mutants of CAP defective in etal.,1996). transcription activation at class II CAP-dependent In the structure of the CAP-DNA complex, the promoters, but not defective in transcription acti- two functional determinants critical for transcrip- vation at class I CAP-dependent promoters, DNA tion activation at class II CAP-dependent promo- binding,andDNAbending(Niuetal.,1996).In ters, i.e. AR1 in the upstream subunit of the CAP the structure of the CAP-DNA complex, the dimer and AR2 in the downstream subunit of the residues that comprise AR2 form a promin- CAP dimer, map to the same face of the CAP ently exposed surface with dimensions of dimer, but are separated by nearly the full length 8AÊ Â20 AÊ . AR2 carries a net positive charge ofthisface(Figure1(b)). of 2, and mutational studies indicate that net RNAP determinants {Tworeportsintheliteratureincorrectlyconclude Class II CAP-dependent transcription requires that aCTD plays no positive role in class II CAP- two sets of determinants in the RNAP a subunit: dependenttranscription(Igarashietal.,1991;Westetal., (i) Class II CAP-dependent transcription, like class 1993).Theincorrectconclusionsinthesereportsare I CAP-dependent transcription, requires aCTD (in attributable to errors in normalization of speci®c this case, the 265 and 287 determinants of aCTD activities of wild-type and mutant RNAP preparations (Figure2;Saveryetal.,1998)){.Experimentswith (D. West & S. B., unpublished results). RNAP derivatives lacking aCTD and properly normalized with oriented-a RNAP derivatives having one full- respect to speci®c activity exhibit defects in class II length a subunit and one truncated a subunit lack- CAP-dependent transcription (D. West & S.B., ing aCTD indicate that only one of the two copies unpublished results; W. Niu & R.H.E., unpublished of aCTD in RNAP is required for class II CAP- results). dependent transcription, and that this copy can be Transcription Activation by CAP 205 either aCTDI (aCTD of the a subunit that interacts sequencewithinthe35-elementregionofaclass with b) or, less favorably, aCTDII (aCTD of the a IICAP-dependentpromoter(Rhodiusetal.,1997). subunit that interacts with b0)(W.N. & R.H.E., unpublished results). Mechanism (ii) Class II CAP-dependent transcription requires a class-II-speci®c determinant within Protein-protein photocrosslinking indicates that, aNTD(Niuetal.,1996).Thisdeterminant,which in the ternary complex of CAP, RNAP, and a class consists of residues 162-165 within aNTD, was II CAP-dependent promoter, AR1 of CAP is in de®ned by the isolation of single amino acid sub- direct physical proximity to aCTD of RNAP (Y. stitution mutants defective in class II CAP-depen- Chen, W. Niu & R.H.E., unpublished results), and dent transcription, but not defective in class I CAP- AR2 of CAP is in direct physical proximity to the dependenttranscriptionorCAP-independenttran- aNTDofRNAP(Niuetal.,1996).DNAaf®nity scription(Niuetal.,1996).Inthestructureof cleaving with RNAP derivatives having EDTA:Fe aNTD, the residues that comprise this determinant incorporated within aCTD indicates that, in the are located within a prominently accessible surface complex, aCTD is in direct physical proximity to loop(Zhang&Darst,1998).Allfourresiduesthat the DNA segment immediately upstream of the comprise this determinant are negatively charged, DNAsiteforCAP(Murakamietal.,1997). and net negative charge appears to be critical for Together with the above-described results de®ning functionofthedeterminant(Niuetal.,1996).Pre- critical determinants of CAP, RNAP, and promoter liminary experiments with oriented-a RNAP DNA, the photocrosslinking and af®nity cleaving derivatives carrying one wild-type and one mutant results lead to the proposal that CAP activates a subunit indicate that this determinant is function- transcription at class II promoters through two dis- ally presented in only one of the two a subunits, tinctsetsofinteractions(Figure4): i.e. aI, the a subunit that interacts with b (W. Niu (i) AR1 of the upstream subunit of the CAP & R.H.E., unpublished results). dimer is proposed to interact with one of the two copies of aCTD of RNAP (either aCTDI or aCTDII, but preferentially aCTDI), facilitating binding of DNA determinants that copy of aCTD to the DNA segment immediately upstream of CAP. The interaction with AR1 Hydroxyl radical and DNase I DNA footprinting is proposed to involve the 287 determinant of experiments indicate that formation of the ternary aCTD, and the interaction with DNA is proposed complex of CAP, RNAP and a class II CAP-depen- to involve the 265 determinant of aCTD. dent promoter results in protection, not only of the (ii) AR2 of the downstream subunit of CAP is DNA site for CAP and the core promoter, but also proposed to interact with aNTDI, interacting with of the DNA segment immediately upstream of the residues 162-165 within aNTDI. DNAsiteforCAP(Atteyetal.,1994;Belyaevaetal., The two sets of interactions have separate and 1996,1998).FullprotectionofthisDNAsegment distinct mechanistic consequences for transcription requires the integrity of both AR1 of CAP and activation(Niuetal.,1996;Rhodiusetal.,1997): aCTD. At the best-characterized class II CAP- (i) The interaction between AR1 and aCTD dependent promoter, CC(41.5), there is no increases the binding constant, KB, for the for- speci®c DNA sequence determinant in this DNA mation of the RNAP-promoter closed complex and segment. Nevertheless, replacement of this DNA has no effect on the rate constant, kf, for sub- segment by a high-af®nity DNA site for aCTD, sequent isomerization of the closed complex to the such as an UP-element or an UP-element subsite, opencomplex(Niuetal.,1996;Rhodiusetal., resultsinanincreaseintranscription(Lloydetal., 1997).TheincreaseinKB arises from two sources. 1998).Insuchcases,theoptimalspacingbetween First, and most obvious, the AR1-aCTD interaction the DNA site for CAP and the UP-element or UP- directly increases the af®nity of RNAP for promo- element subsite appears to be four base-pairs ter DNA (W.N. & R.H.E., unpublished results; D. (Lloydetal.,1998;G.Lloyd,W.Niu,R.H.E.&S.B., West & S.B., unpublished results). Second, the unpublished results). AR1-aCTD interaction compensates the energetic At a class II CAP-dependent promoter, the pre- cost of displacing aCTD from its preferred location sence of a consensus DNA site for CAP overlap- on promoter DNA and positioning aCTD at a less ping the 35 element precludes the presence of a preferred location on promoter DNA (``anti-inhi- consensus 35 element. DNA af®nity cleaving bition'';Busby&Ebright,1997),anenergeticcost experiments with RNAP derivatives having imposed by the fact that aCTD prefers to interact EDTA:Fe incorporated within s70 region 4 indicate with DNA in the 42 region (N. Naryshkin, A. that s70 region 4 interacts with the non-consensus Revyakin, Y. Kim, H. Chen, H. Tang & R.H.E., 35 element of a class II CAP-dependent promoter unpublished) and the fact that, at class II CAP- in a manner largely similar to that in which it dependent promoters, CAP binds to DNA in the interacts with the consensus 35 element of a con- 42 region, necessitating displacement of aCTD. sensus promoter (J. Bown, A. Kolb, A. Ishihama & (ii) The interaction between AR2 and aNTD, S.B., unpublished results). Consistent with this in contrast to the interaction between AR1 and ®nding, transcription initiation is sensitive to DNA aCTD, functions at a step subsequent to the 206 Transcription Activation by CAP

Figure 4. Transcription activation at class II CAP- dependent promoters, e.g. galP1, CC(41.5). Transcription activation at class II CAP-dependent promoters involves two CAP-RNAP interactions: (i) interaction between AR1 of the upstream subunit of CAP (®lled circle) and the 287 determinant of one copy of aCTD, an interaction that facilitates binding of aCTD, through its 265 determinant to the DNA segment immediately upstream of CAP; and (ii) interaction between AR2 of the downstream subunit of CAP (not visible in this orientation; located directly beneath the N in aNTD) and residues 162-165 of one copy of aNTD. The location of the second, non- contacted copy of aCTD has not been determined de®nitively; therefore, the second copy of aCTD is drawn arbitra- rilybehindthe®rst.AvailableevidencesuggeststhatthesecondcopyofaCTDmayinteract,throughits265determi- nant,withtheDNAsegmentupstreamofthe®rstcopyofaCTD(Belyaevaetal.,1996,1998;Murakamietal.,1997; Lloydetal.,1998).AdaptedfromNiuetal.(1996).

initialbindingofRNAPtopromoterDNA(Niu A third, non-native CAP-RNAP interaction at class etal.,1996;Rhodiusetal.,1997).Thus,thisinter- II CAP-dependent promoters action does not affect the binding constant, KB, for formation of the RNAP-promoter closed com- AR1 and AR2 can be supplemented by a third, non-native activating region (residues 52-58; ``acti- plex, but, rather, increases the rate constant, k , f vatingregion3``,AR3;Belletal.,1990;Williams for isomerization of closed complex to open etal.,1991;Westetal.,1993;Niuetal.,1996).AR3 complex. The mechanism by which the AR2- is created by substitution of Lys52 by a neutral or aNTD interaction facilitates isomerization is not negatively charged residue. Substitution of Lys52 known. Based on the structure of the CAP-DNA substantially increases transcription activation at complex and the position of the DNA site for class II CAP-dependent promoters, but not class I CAP at class II promoters, the AR2-aNTD inter- CAP-dependent promoters, and at least partly sup- action is expected to take place more than 30 AÊ presses effects of substitutions in AR1, AR2, or from the DNA and more than 70 AÊ from the both(Belletal.,1990;Williamsetal.,1991;West RNAPactivesite(Niuetal.,1996).Inprinciple, etal.,1993;V.Rhodius&S.B,unpublishedresults). twomechanismsarepossible:theAR2-aNTD In addition, substitution of Lys52 substantially interaction may trigger an allosteric change strengthens the interaction in solution between between an inactive RNAP conformation and an CAP-DNAbinarycomplexesandRNAP(Niuetal., active RNAP conformation, or the AR2-aNTD 1996).WesuggestthatsubstitutionofLys52exerts interaction may selectively stabilize the transition these effects by creating a non-native energetically statebetweenclosedcomplexandopencomplex favorable interaction with RNAP. (Niuetal.,1996). Alanine scanning of a CAP derivative having a TranscriptionactivationatclassIICAP-depen- functional AR3 (i.e. a CAP derivative with a substi- dent promoters thus provides a paradigm for tution of Lys52) indicates that Glu58 is essential for understanding how a single activator molecule can thefunctionofAR3(Williamsetal.,1991;V.Rho- make multiple interactions with the transcription dius & S.B., unpublished results). In the structure machinery, with each interaction being responsible of the wild-type CAP-DNA complex, Lys52 and for a speci®c mechanistic consequence. Such mul- Glu58 form a salt bridge. We suggest that substi- tiple interactions are likely to be a common feature tution of Lys52 ``unmasks'' the negative charge of of transcription activation. Glu58 and thereby creates a non-native, energetically favorable, electrostatic interaction with Transcription activation at class II CAP-depen- RNAP. Oriented-heterodimer analysis indicates dent promoters involves interactions that take that AR3, when functional, is presented by the place upstream of the DNA site for CAP, and thus downstreamsubunitoftheCAPdimer(Figure1(b); upstream of the locus of CAP-induced DNA bend- Williamsetal.,1996). ing(Figure4).Therefore,itappearslikelythat Several lines of evidence indicate that AR3, CAP-induced DNA bending plays a role in tran- when functional, interacts with s70 region 4, scription activation at class II CAP-dependent pro- speci®cally with residues 590-600, which immedi- moters, i.e. facilitation of upstream CAP-aCTD and ately follow the helix-turn-helix motif responsible aCTD-DNA interactions. Consistent with this infer- for recognition of the promoter 35 element. First, ence, substitutions in CAP that result in a decrease substitution of Lys593, Lys597, or Arg599 of s70 in CAP-induced bending result in a measurable reduces or eliminates AR3-dependent transcription (albeit modest) defect in transcription activation at activation at class II CAP-dependent promoters classIICAP-dependentpromoters(A.Kapanidis& (Lonettoetal.,1998).Second,site-speci®cprotein- R.H.E., unpublished). protein photocrosslinking indicates that AR3 is in Transcription Activation by CAP 207 direct physical proximity to s70 in the ternary com- Synergistic transcription activation by CAP and plex of CAP, RNAP, and a class II CAP-dependent other activators promoter(Jinetal.,1995).Third,modelbuilding suggests that AR3 of the downstream subunit of At many CAP-dependent promoters, CAP syner- CAP is in direct physical proximity to residues gistically activates transcription with one or more 590-600ofs70intheternarycomplex(Busby& Ebright,1997;Lonettoetal.,1998).Fourth,AR3, other activators. when active, carries a net negative charge and At some promoters where CAP synergistically therefore is electrostatically complementary to resi- activates transcription with a second, different acti- dues 590-600 of s70, which carry a net positive vator, the mechanisms of transcription activation charge. are similar to the mechanisms described in the pre- The AR3-s70 interaction has no effect on the ceding section. Thus, a CAP dimer centered near position 103 binding constant, KB, for formation of the RNAP- promoter closed complex, and affects only the rate or position 93 can synergistically activate tran- constant, k , for isomerization of closed complex to scription with a second, different activator able to f 70 the open complex (V. Rhodius & S.B., unpublished interact with aCTD, aNTD, and/or s (e.g. lcI or results). Interestingly, lcI, another activator that FNRcenterednearposition42;Joungetal.,1994; interactswithresidues590-600ofs70(Lietal., Scottetal.,1995).Insuchcases,CAPfunctionsby 1994),andthatiselectrostaticallycomplementary a class I mechanism, with AR1 of the downstream toresidues590-600ofs70(Bushmanetal.,1989), subunit of CAP interacting with one copy of likewise affects only k (Hawley&McClure,1982). aCTD, and the second activator interacts with the f other copy of aCTD, aNTD, and/or s70 (cf. Figure5(b)). Similarly, a CAP dimer centered near position Transcription activation at class III 42 can synergistically activate transcription with CAP-dependent promoters a second, different activator able to interact with aCTD(e.g.FNRcenterednearposition103,pos- ition93,orposition83;Busbyetal.,1994; Saveryetal.,1996).Insuchcases,CAPfunctions Synergistic transcription activation by multiple through a class II mechanism, with AR1, AR2, and, CAP dimers if present, AR3, interacting with, respectively, one 70 At some CAP-dependent promoters, two or copy of aCTD, aNTD, and s ; and the second acti- more CAP dimers synergistically activate transcrip- vator interacts with the other copy of aCTD (cf. tion. Such promoters have diverse architectures, Figure5(b)). with different distances between the two DNA In each of these cases, synergistic transcription sites for CAP, and different distances between the activation results from the fact that CAP and the DNA sites for CAP and the DNA site for RNAP. second, different activator make independent con- Remarkably, despite the apparently complex, tacts with different surfaces of RNAP (and possibly diverse architectures of these promoters, transcrip- also affect different steps in transcription tion activation at such promoters is relatively initiation). Importantly, these mechanisms for simple, involving straightforward, additive com- synergistic transcription activation do not require binations of the elementary class I and class II direct interaction between CAP and the second mechanisms. activator, and thus it is possible for CAP to syner- Thus, a CAP dimer centered near position 103 gistically activate transcription through these or position 93 can synergistically activate tran- mechanisms with a broad range of unrelated acti- scription with a CAP dimer centered near position vators. 62(Joungetal.,1993;Lawetal.,1999;Langdon& At other promoters where CAP functions Hochschild,1999).Insuchcases,eachCAPdimer together with a second, different activator, the functionsthroughaclassImechanism,withAR1 mechanisms of transcription activation are more of the downstream subunit of each CAP dimer complex. Thus, at some promoters, CAP functions, interactingwithonecopyofaCTD(Figure5(a)). at least in part, through direct protein-protein Similarly, a CAP dimer centered near position interaction with a second activator that facilitates 103, position 93, or position 83 can synergisti- interactions between the second activator and cally activate transcription with a CAP dimer cen- DNA, through CAP-induced DNA bending that terednearposition42(Busbyetal.,1994; facilitates interactions between a second activator Murakamietal.,1997;Belyaevaetal.,1998).In and RNAP, and/or through CAP-induced DNA such cases, the upstream CAP dimer functions by bending that disrupts inhibitory interactions a class I mechanism, with AR1 of the downstream (Lobell&Schleif,1991;Richetetal.,1991;Forsman subunit interacting with one copy of aCTD; and etal.,1992;Perez-Martin&Espinosa,1993;Merkel the downstream CAP dimer functions by a class II etal.,1995).Atsuchpromoters,theAR1-aCTD, mechanism, with AR1, AR2, and, if present, AR3, AR2-aNTD, and AR3-s70 interactions critical for interacting with, respectively, the other copy of transcription activation at class I and class II CAP- aCTD,aNTD,ands70region4(Figure5(b)). dependent promoters play little or no role. 208 Transcription Activation by CAP

Figure 5. Transcription activation and anti-activation at class III CAP- dependent promoters. (a) Quatern- ary complex of two CAP dimers, RNAP, and a class III CAP-dependent promoter having one DNA site for CAP centered near position 103 or position 93, and a second DNA site for CAP centered near position 62. Each CAP dimer functions through a class I mechanism, with AR1 of the downstream subunit of each CAP dimer (open, dashed circle) interacting with one copyofaCTD(cf.Figure3). (AdaptedfromLangdon& Hochschild,1999).(b)Quaternary complex of two CAP dimers, RNAP, and a class III CAP-dependent promoter having one DNA site for CAP centered near position 103, position 93, or position 83, and a second DNA site for CAPcenterednearposition42.TheupstreamCAPdimerfunctionsbyaclassImechanism,withAR1ofthedown- streamsubunit(open,dashedcircle)interactingwithonecopyofaCTD(cf.Figure3(b));thedownstreamCAPdimer functions by a class II mechanism, with AR1 (®lled circle), AR2 (not visible in this orientation; located directly beneath the N in aNTD), and, if present, AR3, interacting with, respectively, the other copy of aCTD, aNTD, and s70 region4(cf.Figure4).(AdaptedfromMurakamietal.,1997;Belyaevaetal.,1998.)(c)TernarycomplexofCytR,two CAP dimers, and a CytR-regulated class III CAP-dependent promoter (``anti-activation complex''). CytR makes protein-protein interactions with the two CAP dimers, interacting with residues 12, 13, 17, 105, 108 and 110 of the CytR-proximal subunit of each CAP dimer (squares), and protein-DNA interactions with the DNA segment between the two CAP dimers. The CytR-CAP and CytR-DNA interactions block transcription activation by CAP (anti-activate) by blocking the functional AR1 of each CAP dimer and by preventing interaction of aCTD with the DNA segment adjacenttoeachCAPdimer(cf.(b)).(AdaptedfromValentin-Hansenetal.,1996;Kallipolitisetal.,1997.)

Anti-activation amino acid substitutions that render CAP insensi- tive to anti-activation by CytR, but that do not Anti-activation by blocking CAP or DNA interfere with transcription activation, DNA bind- determinants: CytR ing,orDNAbendingbyCAP(Sùgaard-Andersen etal.,1991;Meibometal.1999).Thedeterminant CytR uses an ``anti-activation'' mechanism to consists of residues Glu12, Trp13, His17, Leu105, inhibit transcription initiation at a subset of CAP- Val108, and Pro110 in the N-terminal, cAMP-bind- dependent promoters involved in pyrimidine ing domain of CAP. Oriented-heterodimer analysis metabolism, most notably the deoP2, udp, nupG, indicates that, for each of the two CAP dimers, the andcddpromoters(Mollegaardetal.,1993; determinant is functionally presented in the CAP Valentin-Hansenetal.,1996).Eachofthesepromo- subunit proximal to the DNA site for CytR (K. ters has a DNA site for CAP centered near position Meibom, B. Kallipolitis, P. Valentin-Hansen & 94 and a second DNA site for CAP centered near R.H.E., unpublished results). position 42 (as in the class III CAP-dependent The determinant of CytR that mediates CytR- promotersinFigure5(b)).Intheabsenceofthe CAP interaction also has been identi®ed, and a allosteric effector cytidine, CytR inhibits transcrip- detailed model for the structural organization of tion initiation at these promoters by making pro- the (CAP)2-CytR-DNA anti-activation complex has tein-protein interactions with the two CAP dimers beenproposed(Kallipolitisetal.,1997). bound to promoter DNA and protein-DNA interactions with the DNA segment between the two Anti-activation by blocking RNAP determinants: CAPdimers(Figure5(c)).TheCytR-CAPand bacteriophage T4 ADP-ribosylation CytR-DNA interactions completely block transcription activation by CAP, by sterically blocking the Interference with transcription activation by functional AR1 of each CAP dimer and by steri- CAP can also be accomplished by blocking essen- cally preventing aCTD from interacting with the tial determinants on RNAP. During infection of DNA segment adjacent to each CAP dimer (cf. E. coli by bacteriophage T4, the T4 alt and mod Figures5(b)and(c)). gene products ADP-ribosylate Arg265 of RNAP a The determinant of CAP involved in CytR-CAP subunit(Goff,1984),themostcriticalresidueofthe interaction has been identi®ed by isolation of single 265determinantofaCTD(Gaaletal,1996; Transcription Activation by CAP 209

Murakamietal.,1996;Saveryetal.,1998).ADP- Acknowledgments ribosylation of Arg265 blocks aCTD-DNA interaction and thus selectively abolishes CAP-depen- We thank S. Darst, R. Gourse, A. Hochschild, W. dent transcription and UP-element-dependent Ross, P. Valentin-Hansen, and current and past mem- transcription, without affecting CAP-independent, bers of our laboratories for access to unpublished results. Work in our laboratories on transcription acti- UP-element-independent transcription (K. Severi- vation by CAP has been supported by project grants nov, W. Ross, H. Tang, L. Snyder, A. Goldfarb, R. from the UK BBSRC and the Wellcome Trust to S.B., Gourse & R.H.E., unpublished results). and by a Howard Hughes Medical Institute Investiga- torship and National Institutes of Health grant GM41376 to R.H.E.

Implications The mechanisms summarized here for transcrip- References tion activation by CAP can be generalized to other Attey, A., Belyaeva, T., Savery, N., Hoggett, J., Fujita, bacterial activators. N., Ishihama, A. & Busby, S. (1994). Interactions In particular, these mechanisms apply with full between the cyclic AMP receptor protein and the force to FNR, a distant sequence and structural alpha subunit of RNA polymerase at the E. coli homologofCAP(Guestetal.,1996).FNRactivates galP1 promoter. Nucl. Acids Res. 22, 4375-4380. transcription at promoters organized precisely ana- Beckwith, J., Grodzicker, T. & Arditti, R. (1972). Evi- logously to class I, class II, and class III CAP- dence for two sites in the lac promoter region. dependentpromoters(Wingetal.,1995;Scottetal., J. Mol. Biol. 69, 155-160. 1995).FNRcontainsfunctionalcounterpartsof Bell, A., Gaston, K., Williams, R., Chapman, K., Kolb, AR1 and AR3, and FNR activates transcription A., Buc, H., Minchin, S., Williams, J. & Busby, S. 70 (1990). Mutations that affect the ability of the through AR1-aCTD and AR3-s interactions that, Escherichia coli cyclic AMP receptor protein to acti- in all signi®cant respects, are equivalent to the vate transcription. Nucl. Acids Res. 17, 3865-3874. interactions made by CAP and summarized in Belyaeva, T., Bown, J., Fujita, N., Ishihama, A. & Busby, Figures3and4(Williamsetal.,1997;Lietal.,1998; S. (1996). Location of the C-terminal domain of the Lonettoetal.,1998). RNA polymerase alpha subunit in different open A large subset of bacterial activators unrelated in complexes at the E. coli galactose operon regulatory sequence and structure to CAP also appears to region. Nucl. Acids Res. 24, 2243-2251. activate transcription by interacting with promoter Belyaeva, T., Rhodius, V., Webster, C. & Busby, S. DNA upstream of the 35 element and making (1998). Transcription activation at promoters carry- protein-protein interactions with aCTD that, in all ing tandem DNA sites for the Escherichia coli cyclic AMP receptor protein: organization of the RNA signi®cant respects, are equivalent to those made polymerase a subunits. J. Mol. Biol. 277, 789-804. by CAP at class I CAP-dependent promoters and Blatter, E., Ross, W., Tang, H., Gourse, R. & Ebright, R. summarizedinFigure3(Busby&Ebright,1994; (1994). Domain organization of RNA polymerase Ebright&Busby,1995;Rhodius&Busby,1998). alpha subunit: C-terminal 85 amino acids constitute Another large subset of bacterial activators appears a domain capable of dimerization and DNA bind- to function by interacting with promoter DNA in ing. Cell, 78, 889-896. the 35 element region and making concurrent Brennan, R. (1991). Interactions of the helix-turn-helix protein-protein interactions with aCTD, aNTD, binding domain. Curr. Opin. Struct. Biol. 1, 80-88. and/or s70 analogous to those made by CAP at Brennan, R. (1992). DNA recognition by the helix-turn- class II CAP-dependent promoters and summar- helix motif. Curr. Opin. Struct. Biol. 2, 100-108. Burgess, R. (1976). Puri®cation and physical properties izedinFigure4(Busby&Ebright,1994;Rhodius of E. coli RNA polymerase. In RNA Polymerase &Busby,1998). (Losick, R. & Chamberlin, M., eds), pp. 69-100, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Busby, S. & Ebright, R. (1994). Promoter structure, pro- Prospect moter recognition, and transcription activation in prokaryotes. Cell, 79, 743-746. Transcription activation at class I, class II, and Busby, S. & Ebright, R. (1997). Transcription activation the simplest class III CAP-dependent promoters at class II CAP-dependent promoters. Mol. Micro- should be amenable to a complete structural and biol. 23, 853-859. mechanistic description. Priorities for future work Busby, S., West, D., Lawes, M., Webster, C., Ishihama, include elucidation of the structures of the AR1- A. & Kolb, A. (1994). Transcription activation by aCTD, AR2-aNTD, and AR3-s70 interfaces; deter- the E. coli cyclic AMP receptor protein: receptors mination of when CAP-RNAP interactions are ®rst bound in tandem at promoters can interact syner- made on the pathway from free promoter to gistically. J. Mol. Biol. 241, 341-352. Bushman, F., Shang, C. & Ptashne, M. (1989). A single RNAP-promoter closed complex, to RNAP-promo- glutamic acid residue plays a key role in the tran- ter open complex; and determination of when, and scriptional activation function of lambda repressor. how, these interactions are broken in promoter Cell, 58, 1163-1171. escape. Methods to address these priorities are in Chamberlin, M. (1976). RNA polymerase - an overview. place. Progress should be rapid. In RNA Polymerase (Losick, R. & Chamberlin, 210 Transcription Activation by CAP

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