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Identification of a Surface of FNR Overlapping Activating Region 1 That Is Required for Repression of Gene Expression

Identification of a Surface of FNR Overlapping Activating Region 1 That Is Required for... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 15, Issue of April 9, pp. 10244 –10248, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Identification of a Surface of FNR Overlapping Activating Region 1 That Is Required for Repression of Gene Expression* (Received for publication, July 21, 1998, and in revised form, December 7, 1998) Jeffrey Green‡ and Fiona A. Marshall From the Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom A library of Escherichia coli fnr mutants has been RNA polymerase to activate transcription (2). At promoters in screened to identify FNR (regulator of fumarate and which the FNR site is centered at 261 or beyond (Class I) nitrate reduction) variants that are defective repres- activation depends on a different AR-a subunit contact (3). sors, but competent activators. All but one of seventeen As well as acting as an activator of anaerobic gene expres- variants had substitutions close to or within the face of sion, FNR also acts as a repressor of some aerobic genes (1). FNR that contains activating region 1 (AR1). Activating Unlike the FNR-activated promoters, there is no discernible a subunit of RNA po- region 1 is known to contact the pattern of FNR site positioning among FNR-repressed promot- lymerase to facilitate transcription activation. It is now ers, although a common feature is the presence of multiple evident that this face also has a role in FNR-mediated FNR sites upstream of the transcription start (1). The best repression. Single amino acid substitutions at Lys characterized of the FNR-repressed genes is ndh, which en- 74 95 147 193 197 239 , Ala , Met , Leu , Arg ,orLeu , and double Gly codes a non-proton-translocating NADH dehydrogenase that is 13 145 16 45 69 and Ser , Cys and Ile , Tyr substitutions at Ser the major primary dehydrogenase of the aerobic respiratory 133 164 191 and Ser ,orLys and Phe , impaired FNR-medi- chain (4). The ndh promoter has two FNR sites centered at ated repression of ndh without greatly affecting activa- 250.5 and 294.5, and both contribute to FNR-mediated repres- tion from model Class I (FNR site at 271.5) and Class II sion (5–7). The mechanism of FNR-mediated repression ap- (FNR site at 241.5) FNR-activated promoters. Although pears not to be due to simple promoter occlusion, but rather repression was impaired in a second group of FNR vari- 34 72 193 92 displacement of the RNA polymerase a subunit leads to inhi- , Arg and Leu , Phe , ants with substitutions at Leu bition of transcription (7). FNR sites located beyond the region , transcription activation from the simple FNR- or Ser of DNA occupied by RNA polymerase have also been shown to dependent promoters was severely reduced. However, be necessary for efficient repression of the fnr and narX pro- expression from pyfiD (FNR sites at 240.5 and 293.5) moters (8). Furthermore, FNR occupation of the far upstream and a derivative lacking the site at 293.5, pyfiD2/1, remained relatively high indicating that this second FNR site of the yfiD promoter (FNR sites at 240.5 and 293.5) group have a context-dependent activation defect as down-regulates yfiD expression (9). These observations indi- well as a repression defect. The prediction that the sub- cated that FNR-mediated repression of these promoters re- stitutions affecting repression were likely to be in sol- quires multiple FNR binding sites and thus repression may vent exposed regions of FNR was supported by analysis arise a consequence of interactions between two or more FNR of peptides produced by partial proteolysis of FNR. dimers, or between FNR dimers (or tetramers) and RNA po- Thus, FNR-mediated repression at promoters with mul- lymerase. Therefore a library of fnr mutants was screened for tiple FNR sites requires regions of FNR that are differ- FNR variants defective in repression. Two types of variant ent from, but overlap, AR1. were identified: type A was a poor repressor but good activator; type B was a poor repressor but failed to activate transcription from simple FNR-activated promoters. However, the type B The FNR protein of Escherichia coli is a global transcription variants did activate transcription from the complex yfiD pro- regulator, controlling the expression of genes in response to moter. All the variants isolated, except one, contained amino oxygen starvation. FNR is predicted to be structurally related acid substitutions overlapping the face of FNR that contains to the cAMP receptor protein and acts mainly as an activator of AR1, indicating that this surface may have a role in repression genes involved in anaerobic energy generation (1). Generally, as well as in activation and anti-inhibition. FNR activates transcription by binding to a site centered at about 241 in target (Class II) promoters where it makes mul- EXPERIMENTAL PROCEDURES tiple direct activating contacts with RNA polymerase involving Error-prone Polymerase Chain Reaction Mutagenesis—Random mu- two discrete activating regions of FNR (Fig. 1A, 2 and 3). tations in the fnr gene carried by pGS24 (a derivative of pBR322 Activating region 1 (AR1) appears to contact the C-terminal containing the fnr gene in a HindIII-BamHI fragment) were introduced using Taq DNA polymerase and the following synthetic primers, as domain of the RNA polymerase a subunit, thereby preventing described previously (2): 59-GCTTATCATCGATAAGCTTCGTGAATA- inhibition of transcription activation caused by an untethered a 70 TTTTGCCGG (fnr coordinates 1–23) and 59-CGTAGAGGATCCAGGC- subunit. Activating region 3 (AR3) contacts the s subunit of TGTACGC (1625–1641), where the unique HindIII and BamHI targets are underlined. Following digestion with HindIII and BamHI, the polymerase chain reaction products were ligated into the corresponding * This work was supported by the Biotechnology and Biological Sci- sites of pBR322. Plasmids were isolated by standard methods, and ences Research Council of the United Kingdom and the Royal Society. mutations in the fnr gene were defined by Applied Biosystems cycle The costs of publication of this article were defrayed in part by the sequencing with the aid of two primers: 59-AAACATATGGTCCCGGA- payment of page charges. This article must therefore be hereby marked AAAGCG (520 –536) and 59-GGAAACCTCGATGGTAGCTGAAATCCC- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to GTTCG (866 – 898). indicate this fact. Identification of FNR Variants Defective in Repression—The library ‡ To whom correspondence should be addressed. Tel.: 44-114-222- 4403; Fax: 44-114-272-8697; E-mail: [email protected]. of mutagenized fnr genes in pBR322 was used to transform JRG3701, a 10244 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. FNR-mediated Repression 10245 derivative of JRG1728 [D(lacIPOZYA)X74 galU galK rpsL D(ara-leu) were produced in normal amounts, as judged by Western blot- D(tyrR-fnr-rac-trg)17 zdd-230::Tn9] containing a compatible yfiD-lac ting (Table I). Expression of fnr is autoregulated, partially by reporter plasmid, pGS1000 (10). Transformants were tested for en- binding at a site overlapping the transcript start such that hanced yfiD-lacZ expression on L-agar containing: 5-bromo-4-chloro-3- FNR acts as a molecular brick. Hence, the amount of each FNR indoyl b-galactoside (40 mg/ml), ampicillin (200 mg/ml), and tetracycline variant may reflect their relative affinities for this site as well (35 mg/ml). The plates were incubated under anaerobic conditions at 37 °C for 16 h, after which they were exposed to air. The colonies were as any effects that the particular substitutions may have on monitored and those that became blue first were noted and recovered. protein stability. Even those produced at lower levels were The levels of yfiD-lacZ expression were determined by measuring b-ga- expressed well enough for the screening protocol, because, al- lactosidase activity (11) with cultures that had been grown anaerobi- though chromosomal expression yielded least FNR (,5% of cally for 16 h at 37 °C in sealed bottles containing L broth supplemented that obtained with pGS24), it was still sufficient to allow reg- with glucose (0.4% w/v), ampicillin (200 mg/ml), and tetracycline (35 mg/ml). The pBR322 derivatives containing the mutant fnr genes were ulation of yfiD expression comparable with that observed with isolated and were then used to transform JRG3917, a JRG1728 deriv- multicopy fnr (Table I). This reflects the relative abundance of ative containing a pRW2-based ndh-lacZ reporter, pndh (2), and the the reporter plasmid (2–5 copies per cell) compared with the degree of repression conferred by the FNR variants was estimated by fnr-encoding plasmid (15–20 copies per cell). Therefore, the measuring b-galactosidase activity as above. As anaerobic ndh-lacZ failure to down-regulate yfiD is probably not due to a lack of expression of fnr strains is enhanced during growth on rich medium (6), FNR in the cell. the effect of the FNR variants on ndh-lacZ expression was also deter- Nucleotide sequence analysis revealed that most of the FNR mined following growth on a glucose minimal medium supplemented with leucine (12) and appropriate antibiotics. Similarly, two simple variants encoded by the plasmids contained substitutions in FNR-activated promoters, the Class I FF-71.5pmelR (FNR site at the face of FNR that contains AR1 (Table I, Fig. 1A). The only 271.5) and Class II FFpmelR (FNR site at 241.5), as well as pyfiD exception was the variant M147T (which was isolated twice) promoter mutants with impaired FNR sites at either 293.5 (pyfiD2/1; representing a replacement buried in the dimer interface (helix pGS1062) or 240.5 (pyfiD1/2; pGS1063) fused to lac in pRW50 were a ). There is no obvious reason why such a substitution should used to determine the effects of the selected amino acid substitutions on FNR-mediated activation (9, 13). Western blotting of the soluble frac- result in the properties observed; however, it should be noted tion of sonic cell-free extracts with polyclonal anti-FNR serum has been that the M147T variant still retained significant repressing described previously (14). The relative amount of each FNR variant was ability (Table I). Many of the substitutions (K54E, Y69C, R72L, estimated by quantitative densitometry using a Vilber-Lourmat imag- G74C, F92S, A95P) were clustered in a series of loops that form ing system. the AR1 side of the FNR b-roll. A second cluster was evident in Partial Proteolysis—The FNR protein was purified from a glutathi- the region encompassing a to a (S178F, F191L, L193P, one S-transferase-FNR fusion protein as described (15). Aliquots (20 ml, D E 3.4 mg/ml) of the isolated FNR were incubated at 30 °C for up to2hin R197H). Four variants (G74C, F92S, A95P, and L193P) had the presence of either 0.68 unit of trypsin (Sigma), 1 unit of chymotryp- been identified in a previous screen, and it was suggested that sin (Sigma), or 0.1 unit of V8 protease (Sigma). The peptides generated the defect in yfiD down-regulation was due to an altered AR1- were then fractionated by SDS-polyacrylamide gel electrophoresis and containing surface in these variants (9). Five of the 17 variants transferred to nitrocellulose membranes for N-terminal amino acid identified contained two substitutions, but in each case at least sequencing. one of the replacements was of a residue predicted to be close to RESULTS or part of the AR1-containing surface. The pBR322 derivatives encoding FNR variants defective in Identification of Repression Defective FNR Variants—Previ- yfiD down-regulation were transferred into JRG3917 (Dfnr ous attempts to isolate repression defective FNR variants using Dlac, containing a compatible FNR-repressed ndh-lacZ re- the FNR-repressible ndh promoter as a screen were unsuccess- porter, pndh; Ref. 2). All the variants failed to repress ndh ful, yielding only FNR proteins with reduced affinity for their expression normally indicating that the yfiD and ndh promot- DNA target and therefore compromised in both activation and ers may share a common mechanism of FNR-mediated repres- repression (16). However, recent analysis of the regulation of sion (Table I). As anaerobic expression of ndh is known to yfiD expression in E. coli (9) has provided an opportunity to respond to nutrient quality in fnr strains (6), ndh-lacZ expres- develop a better screen. The yfiD gene has an unusual pro- sion was also determined for cultures grown in a defined min- moter architecture for an FNR-activated gene, with two FNR imal medium (Table I). The data obtained confirmed the FNR sites centered at 240.5 and 293.5 relative to the transcript repression defects observed in rich medium. Tests with equiv- start. Multiple FNR sites are usually found in FNR-repressed alent strains carrying the FNR-activated Class II FFpmelR- promoters; for example, the ndh gene has FNR sites centered lacZ reporter plasmid, which should be substantially unaf- at 250.5 and 294.5 (1). Expression of yfiD is dependent upon fected by substitutions in AR1, indicated that all but four of the an activating FNR dimer centered at 240.5, but occupation of seventeen variants were capable of activating transcription. It the upstream FNR site (293.5) down-regulates expression (9). was expected that all the variants would be competent activa- Therefore, screening a library of randomly mutagenized fnr tors of Class II promoters, because the basis of the original genes for those that allow increased yfiD-lacZ expression (i.e. screen depended on FNR activation from a site at 240.5 in the FNR variants that still activate from position 240.5, but fail to yfiD promoter. Therefore, the response of the simple Class II act as repressors at 293.5) should ensure that the FNR pro- promoter FFpmelR defines two types of FNR variant: Type A, teins selected are not compromised in DNA binding activity. Error-prone polymerase chain reaction mutagenesis was which fails to repress but activates normally, and Type B, which has both repression and activation defects. This assign- used to generate a library of randomly mutated fnr genes in ment was confirmed using a pyfiD2/1 reporter (pGS1062) with pBR322. Transformants of JRG3701 (Dfnr Dlac, contain- ing a compatible yfiD-lacZ reporter plasmid, pGS1000) were an impaired FNR site at 293.5 (Table I). The Type B variants exhibited much reduced activity at this promoter compared screened for elevated yfiD-lacZ expression on 5-bromo-4- chloro-3-indoyl b-galactoside plates. Approximately 4000 colo- with the Type A proteins. Studies with the Class I promoter a contact for activation, nies were screened, and 19 were found to contain plasmids that FF-71.5pmelR, which requires an AR1- enhanced yfiD expression (Table I). Estimation of the b-galac- indicated that the Type A substitutions could be neutral for tosidase activities of anaerobic cultures indicated that all of the (R197H), or improve (K54E, A95P) or impair (I45T, Y69C, FNR variants encoded by the plasmids were defective in the S133P, G74C, M147T, L193P, L239P, L239E), the AR1 contact. down-regulation of yfiD expression. Most of the FNR variants However, any improvement in the AR1 contact was insufficient 10246 FNR-mediated Repression TABLE I Transcription regulation of FNR-repressed and FNR-activated promoters by 17 FNR variants Expression of b-galactosidase driven by the indicated FNR variants was measured in JRG1728 (Dlac Dfnr) transformed with pyfiD,pndh, FFpmelR, or FF-71.5pmelR fused to lac. Values (which varied by no more than 10%) are the mean of duplicate assays of at least two independent anaerobic cultures of each strain, grown on rich medium in sealed bottles at 37 °C for 16 h. Expression of ndh-lacZ was also determined in minimal medium to eliminate the enhancement of ndh expression associated with anaerobic growth on rich medium in the absence of FNR. Aerobic expression from the test promoters for all the variants was similar to that observed for FNR: 100 Miller units for pyfiD; 2100 for pndh; 110 for pyfiD2/1; 150 for FFpmelR; and 150 for FF-71.5pmelR. Expression of each variant was assessed by Western blotting using anti-FNR serum and is given as a percentage (620%) of that observed with cultures expressing fnr from an equivalent plasmid (pGS24, row labeled FNR). The figures in parentheses indicate the number of independent isolates. Anaerobic b-galactosidase activity Codon FNR variant Expression change pndh defined pyfiD pndh pyfiD2/1 FFpmelR FF-71.5pmelR medium % Miller units Dfnr 70 4600 2400 110 70 140 fnr ,5 760 420 6300 2600 FNR 100 680 170 510 8220 6220 3160 Type A: S13P TCT-CCT 100 6750 4790 2300 6710 2440 1090 S145N AGC-AAC I45T ATC-ACC 100 1430 340 800 7180 5790 2830 K54E AAA-GAA 100 3440 510 1120 8160 9790 4580 Y69C TAT-TGT 100 5620 1040 1470 7020 3600 1640 S133P TCC-CCC G74C GGT-TGT 30 5300 4000 1400 7240 5000 650 A95P GCA-CCA 100 2200 1000 1800 5070 7700 4100 M147T (2) ATG-ACG 100 1840 1120 1760 7880 4620 2510 K164E AAA-GAA 100 6750 2710 1840 7450 2730 1000 F191L (2) TTC-CTC L193P CTG-CCG 50 4600 3000 1800 7975 7600 1770 R197H CGT-CAT 100 2100 1130 1660 8110 7160 3020 L239P CTG-CCG 30 4710 1390 1820 9240 4290 1960 L239E CTG-CAG 20 4570 1080 2400 8595 5140 2140 Stop-14 C-terminal amino acid TGA-CGA 20 6470 1680 1250 9450 3680 1670 Type B: L34P CTC-CCC 30 3590 4720 1290 3630 340 290 R72L CGC-CTC 5 6440 4350 1540 5250 880 570 L193P CTG-CCG F92S TTC-TCC 20 5300 6710 2500 1740 285 290 S178F TCC-TTC 100 5640 3900 1990 5910 1110 490 fnr expressed from the chromosome. This variant also contained the substitution C16S, which is known to be neutral for FNR activity. The additional 14 C-terminal amino acids are: RFFRITHYPFCHNH. FIG.1. Positions of amino acid replacements that compromise FNR-mediated repression and locations of solvent exposed regions of FNR. A, predicted structure of an FNR monomer based on that of the cAMP receptor protein showing the positions of amino acid replacements that impair FNR-mediated repression of ndh-lacZ and yfiD-lacZ expression. The helix-turn-helix motif (a -a ) in the DNA-binding domain, the D F essential cysteine residues that act as ligands for the [4Fe,4S] cluster (ringed), and the previously identified activating regions AR1 and AR3 (or 85-loop) are also indicated. B, surface-exposed regions of FNR. Predicted structure of an FNR monomer showing the sites of cleavage by trypsin and V8 protease. to allow activation from a yfiD reporter with an impaired FNR Substitutions Effecting FNR-mediated Repression Are Lo- site at 240.5, pyfiD2/1 (not shown). Furthermore, the variants cated in Surface-exposed Regions—There is no structure avail- G74C and L193P, which activated the Class II promoter well, able for FNR, but there is evidence to indicate that the pre- but were poor at Class I, indicated that the anti-inhibition dicted similarity with the cAMP receptor protein is well contact made between the RNA polymerase a subunit and the founded (1). An essential feature of any regions of FNR in- AR1 face of FNR (required at Class II promoters) is different to volved in contacting other components of the transcription the activating AR1 a subunit contact made at Class I machinery is that they must be solvent exposed. Partial prote- promoters. olysis of isolated FNR with trypsin (28 possible cleavage sites, FNR-mediated Repression 10247 TABLE II Partial proteolysis of FNR Polypeptide products identified following digestion of FNR (68 mg) with trypsin (a) or V8 protease (b). The FNR protein used was FNR-572 released from a glutathione S-transferase-FNR fusion protein (15) as a result, the isolated FNR protein contains an additional 15 N-terminal amino acids (GSPGISGGGGGILDS). Lowercase letters indicate that the amino acid residue was not detected but is from the known se- quence; – indicates that the sequence corresponds to the N-terminal of FNR; the apparent molecular weights (M ) are estimated from compar- isons with standard proteins on the blots. Band and apparent M N-terminal sequences Cleavage position (a) 1 37,000 GSPGIS – 2 36,000 gsPGIsGG – 3 32,000 GSPGISGG – 4 28,000 GSPGISGG – SYTITEQG Lys tITEQG Tyr 5 25,000 SYTITEQGDEQITGF Lys TITEQGDEQITgFHL Tyr 6 22,500 SYTITEQGDEQITGF Lys TITEQGDEQITGF Tyr 7 21,000 HLAGdLVG Phe SYTITEQG Lys TITEQGdE Tyr FIG.2. Positions of amino acid substitutions in FNR defining 8 17,000 SYTITEQGDEQITGF Lys activating regions and repression defective variants superim- TITEEQGDEQITGFHL Tyr posed on the a backbone of a cAMP receptor protein monomer. 9 14,500 GSPGISGGGGGILDS – The position of amino acids forming AR1 (see Refs. 3 and 18) important 72 73 118 120 SYTITEQGDEQITGF Lys in transcription at Class I promoters (Arg , Ser , Thr , Met , 181 184 186 187 188 225 TITEQGdEQITGFHL Tyr Phe , Arg , Phe , Ser , Pro , and Ala ; blue), AR3 (19) re- 81 85 86 10 13,000 GSPGISGGGGGILDS – quired for the activation of Class II promoters (Ile , Gly , Asp , and 112 45 54 69 SYTITEQGDEQITgFH Lys Phe ; green); and repression-defective variants (Ile , Lys , Tyr , 74 92 95 178 193 197 TITEQGDEQITgFH Tyr Gly , Phe , Ala , Ser , Leu , and Arg ; red) are indicated. The 72 191 IIRRiQsggcAiH Arg yellow spheres indicate positions (Arg and Phe ) that are common to GFSPREFRLTMTR Arg AR1 activation and repression. The AR3 defining residues of FNR are (b) clustered on the left (green); the AR1 defining amino acids (blue and 1 33,000 KRIIRRI Glu yellow) form a large surface along the opposite face of FNR to that 2 28,000 LDQLDNIIERKKPIQ Glu containing AR3. The repression-defective mutations encode substitu- 3 17,000 KRIIRRIOsG Glu tions along the AR1 surface (red and yellow residues) and a further TSMVcEIPFE Glu region falling between AR3 and AR1 (red residues only) forming a IPFETLDDS Glu sharks fin-like pattern of substitutions. The repression defective vari- 13 34 239 4 15,500 KRIIRRIQSGG Glu ants with substitutions at Ser , Leu , and Leu lie in regions of FNR TSMVcEIPFETL Glu beyond that encompassed by the cAMP receptor protein structure, 147 133 145 164 Met in the dimer interface, and the Ser , Ser , and Lys (iso- lated as double mutations with partners in the AR1 face) are omitted for 406 possible peptides) or V8 protease (28 possible cleavage clarity. sites, 435 possible peptides) followed by N-terminal amino acid sequencing of the resulting peptides revealed that the substi- direct contact with RNA polymerase to inhibit transcription tutions affecting repression were within regions of FNR acces- initiation (17). The observation that FNR and RNA polymerase sible to the proteases (Table II, Fig. 1B). Following digestion can simultaneously interact with the FNR-repressible ndh pro- with trypsin, separation of peptides by SDS-polyacrylamide gel moter suggested that, in this case, repression is unlikely to be electrophoresis, and blotting, 10 Coomassie Blue-stained bands mediated simply by promoter occlusion (6, 7). The ndh pro- were detected. The N-terminal amino acid sequences of the moter has two FNR sites centered at 250.5 and 294.5, and it peptides present in each band were determined (Table IIa). has been proposed that FNR occupation of these sites prevents Many of the bands contained the same N-terminal sequences, RNA polymerase a subunit from interacting with DNA (7). indicating preferential cleavage at a limited number of posi- Such a repression mechanism may require direct protein con- tions with subsequent C-terminal processing. The preferred 6 77 79 tacts between the two FNR dimers and/or FNR and RNA po- tryptic/chymotryptic cleavage points were: Arg , Lys , Tyr , 92 184 lymerase. Previous attempts to identify FNR variants compro- Phe , and Arg (Fig. 1B). Treatment with V8 protease pro- mised for repression of ndh-lacZ were unsuccessful, because duced four bands with the N-terminal sequences in Table IIb. 4 38 117 the transformants recovered contained plasmids encoding FNR These data imply cleavage at positions Glu , Glu , Glu , and proteins with defects in DNA binding (16). However the yfiD- Glu . Therefore, the region encompassing the series of loops lacZ reporter provided an opportunity to screen out FNR vari- in the AR1 side of the b-roll was particularly susceptible to ants defective in DNA-binding, because this promoter, al- proteolytic cleavage. These results indicate that the regions though down-regulated by FNR occupation of a site centered at identified as important for FNR-mediated repression are sol- 293.5, requires an activating FNR dimer (at 240.5) for vent exposed and also provide further evidence to support the expression. cAMP receptor protein-based predicted FNR structure, because Using yfiD-lacZ as an initial screen 17 FNR variants defec- the equivalent positions are known to be surface exposed in the tive in repressing both yfiD and ndh promoters were identified. cAMP receptor protein. All but one of the variants (M147T) had substitutions near or DISCUSSION within the face of FNR that contains AR1 (Fig. 1, 2), a region of Transcription can be repressed either passively by promoter the protein known to contact the a subunit of RNA polymerase occlusion, i.e. when a regulator blocks access of RNA polymer- and thereby facilitating transcription activation (3). Two types ase to the promoter, or actively, in which the regulator makes of contact can be made depending on the architecture of the 10248 FNR-mediated Repression activated promoter. At Class I promoters (FNR site at or be- domain of the a subunit of RNA polymerase thought to be yond 261) AR1 of the downstream FNR monomer makes an involved in GalR-mediated activation also relieve GalR-medi- activating contact with the a subunit, whereas at Class II ated repression (20). However, the activator complex at galP2 promoters (FNR site at or about 241) AR1 makes an anti- is an open complex, whereas the repressing complex at galP1 is inhibition contact (3). It is now apparent that the same face of a closed complex and thus the context of the GalR-RNA polym- FNR can be involved in repressing transcription at promoters erase contacts are different. These context effects are proposed that contain multiple FNR sites and that the regions involved to be sufficient to allow a single regulatory protein to act as a are solvent exposed. This is supported by the observations that both a repressor and an activator while maintaining similar 72 191 (i) the positions of two substitutions (Arg and Phe , indi- regulator-polymerase contacts (20). Therefore, it is suggested cated in yellow on Fig. 2) are common to FNR variants with that the context in which FNR finds itself at the yfiD and ndh altered AR1 or repression properties; (ii) all the repression promoters favors the formation of a ternary complex incorpo- defective variants (with the exception of the M147T) contain a rating FNR-polymerase contacts that render the complex in- substitution in the AR1 side of the FNR monomer that forms competent for transcription activation. two clusters overlapping the previously defined positions of In conclusion, the FNR variants identified here provide the AR1 (Fig. 2); (iii) most of the repression defective variants first indication that specific regions (amino acids) of FNR that display altered activation from a model Class I (AR1-depend- overlap AR1 may be required for transcription repression at ent) promoter (Table I); and (iv) that several amino acids close promoters with multiple FNR sites. Mutational analysis of the to those substituted in repression defective variants (Leu , C-terminal domain of the RNA polymerase a subunit should 74 92 178 Gly , Phe , Ser ) are sufficiently exposed to allow proteo- determine whether FNR can repress transcription via direct 38 77 92 184 lytic attack (Glu , Lys , Phe , and Arg ). Thus, the sim- contact between the AR1 containing face and RNA polymerase plest explanation for the repression defective phenotype dis- or whether FNR-FNR contacts are the key to FNR-mediated played by the FNR variants is that they possess an altered repression at promoters sharing the ndh architecture. surface that is different from but overlaps AR1 which has a role Acknowledgments—We thank J. R. Guest, S. J. W. Busby, and H. J. in repression of promoters with multiple FNR sites. Wing for many helpful discussions and J. Keen for amino acid It has been suggested recently that transcription regulators sequencing. can be viewed as catalysts (20). 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(1991) Proc. R. Soc. Lond. B Biol. states between the closed and open complex. Indeed, FNR- Sci. 245, 219 –226 mediated inhibition of open complex formation at the ndh 6. Green, J., and Guest, J. R. (1994) Mol. Microbiol. 12, 433– 444 7. Meng, W., Green, J., and Guest, J. R. (1997) Microbiology 143, 1521–1532 promoter has been observed previously (6). The data presented 8. Takahashi, K., Hattori, T., Nakianishi, T., Nohno, T., Fujita, N., Ishihama, A., here suggest that this could be achieved by direct protein- and Taniguchi, S. (1994) FEBS Lett. 340, 59–64 protein contacts involving the face of FNR containing AR1. 9. Green, J., Baldwin, M. L., and Richardson, J. (1998) Mol. Microbiol. 29, 1113–1123 The architecture of a promoter is clearly crucial in determin- 10. Green, J., and Baldwin, M. L. (1997) Mol. Microbiol. 24, 593– 605 ing the effect of a particular regulator (or combination of reg- 11. Miller, J. (1972) Experiments in Molecular Genetics, pp. 352–355, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY ulators) on transcription (21). It is not yet established if the 12. Cole, S. T., and Guest, J. R. (1980) Mol. Gen. Genet. 178, 409 – 418 repression specific components of the AR1 face participate in 13. Wing, H. J., Williams, S. M., and Busby, S. J. W. (1995) J. Bacteriol. 177, FNR-FNR or FNR-RNA polymerase interactions, but it is likely 6704 – 6710 14. Spiro, S., and Guest, J. R. (1987) J. Gen. Microbiol. 133, 3279 –3288 that the various contacts made by the AR1 containing face are 15. Green, J., Irvine, A. S., Meng, W., and Guest, J. R. (1996) Mol. Microbiol. 19, subtly different. Indeed, there is good evidence to suggest that, 125–137 16. Williams, S. M. Wing, H. J., and Busby, S. J. W. (1998) FEMS Microbiol. Lett. for the cAMP receptor protein the anti-inhibition and activat- 163, 203–208 ing contacts made by AR1 are different (22). The GalR protein 17. Choy, H., and Adhya, S. (1996) Escherichia coli and Salmonella typhimurium, is perhaps the best example of an “active” repressor (20). Like American Society of Microbiology Press, Washington, D. C. 18. Ralph, E. T., Guest, J. R., and Green, J. (1998) Proc. Natl. Acad. Sci. U. S. A. FNR, GalR can act either as an activator (of gal promoter 2, 95, 10449 –10452 galP2) or as a repressor (of gal promoter 1, galP1) and a 19. Green, J., and Baldwin, M. L. (1997) Microbiology (Reading) 143, 3785–3793 20. Roy, S., Garges, S., and Adhya, S. (1998) J. Biol. Chem. 273, 14059 –14062 characteristic GalR-RNA polymerase-gal ternary complex is 21. Savery, N., Rhodius, V., and Busby, S. J. W. (1996) Philos. Trans. R. Soc. Lond. formed at each promoter because of putative GalR-RNA polym- B Biol. Sci. 351, 543–550 erase interactions. Mutations in the region of the C-terminal 22. Zhou, Y., Merkel, T. J., and Ebright, R. H. (1994) J. Mol. Biol. 243, 603– 610 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Identification of a Surface of FNR Overlapping Activating Region 1 That Is Required for Repression of Gene Expression

Green, Jeffrey; Marshall, Fiona A.
Journal of Biological Chemistry – Apr 1, 1999
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 15, Issue of April 9, pp. 10244 –10248, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Identification of a Surface of FNR Overlapping Activating Region 1 That Is Required for Repression of Gene Expression* (Received for publication, July 21, 1998, and in revised form, December 7, 1998) Jeffrey Green‡ and Fiona A. Marshall From the Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom A library of Escherichia coli fnr mutants has been RNA polymerase to activate transcription (2). At promoters in screened to identify FNR (regulator of fumarate and which the FNR site is centered at 261 or beyond (Class I) nitrate reduction) variants that are defective repres- activation depends on a different AR-a subunit contact (3). sors, but competent activators. All but one of seventeen As well as acting as an activator of anaerobic gene expres- variants had substitutions close to or within the face of sion, FNR also acts as a repressor of some aerobic genes (1). FNR that contains activating region 1 (AR1). Activating Unlike the FNR-activated promoters, there is no discernible a subunit of RNA po- region 1 is known to contact the pattern of FNR site positioning among FNR-repressed promot- lymerase to facilitate transcription activation. It is now ers, although a common feature is the presence of multiple evident that this face also has a role in FNR-mediated FNR sites upstream of the transcription start (1). The best repression. Single amino acid substitutions at Lys characterized of the FNR-repressed genes is ndh, which en- 74 95 147 193 197 239 , Ala , Met , Leu , Arg ,orLeu , and double Gly codes a non-proton-translocating NADH dehydrogenase that is 13 145 16 45 69 and Ser , Cys and Ile , Tyr substitutions at Ser the major primary dehydrogenase of the aerobic respiratory 133 164 191 and Ser ,orLys and Phe , impaired FNR-medi- chain (4). The ndh promoter has two FNR sites centered at ated repression of ndh without greatly affecting activa- 250.5 and 294.5, and both contribute to FNR-mediated repres- tion from model Class I (FNR site at 271.5) and Class II sion (5–7). The mechanism of FNR-mediated repression ap- (FNR site at 241.5) FNR-activated promoters. Although pears not to be due to simple promoter occlusion, but rather repression was impaired in a second group of FNR vari- 34 72 193 92 displacement of the RNA polymerase a subunit leads to inhi- , Arg and Leu , Phe , ants with substitutions at Leu bition of transcription (7). FNR sites located beyond the region , transcription activation from the simple FNR- or Ser of DNA occupied by RNA polymerase have also been shown to dependent promoters was severely reduced. However, be necessary for efficient repression of the fnr and narX pro- expression from pyfiD (FNR sites at 240.5 and 293.5) moters (8). Furthermore, FNR occupation of the far upstream and a derivative lacking the site at 293.5, pyfiD2/1, remained relatively high indicating that this second FNR site of the yfiD promoter (FNR sites at 240.5 and 293.5) group have a context-dependent activation defect as down-regulates yfiD expression (9). These observations indi- well as a repression defect. The prediction that the sub- cated that FNR-mediated repression of these promoters re- stitutions affecting repression were likely to be in sol- quires multiple FNR binding sites and thus repression may vent exposed regions of FNR was supported by analysis arise a consequence of interactions between two or more FNR of peptides produced by partial proteolysis of FNR. dimers, or between FNR dimers (or tetramers) and RNA po- Thus, FNR-mediated repression at promoters with mul- lymerase. Therefore a library of fnr mutants was screened for tiple FNR sites requires regions of FNR that are differ- FNR variants defective in repression. Two types of variant ent from, but overlap, AR1. were identified: type A was a poor repressor but good activator; type B was a poor repressor but failed to activate transcription from simple FNR-activated promoters. However, the type B The FNR protein of Escherichia coli is a global transcription variants did activate transcription from the complex yfiD pro- regulator, controlling the expression of genes in response to moter. All the variants isolated, except one, contained amino oxygen starvation. FNR is predicted to be structurally related acid substitutions overlapping the face of FNR that contains to the cAMP receptor protein and acts mainly as an activator of AR1, indicating that this surface may have a role in repression genes involved in anaerobic energy generation (1). Generally, as well as in activation and anti-inhibition. FNR activates transcription by binding to a site centered at about 241 in target (Class II) promoters where it makes mul- EXPERIMENTAL PROCEDURES tiple direct activating contacts with RNA polymerase involving Error-prone Polymerase Chain Reaction Mutagenesis—Random mu- two discrete activating regions of FNR (Fig. 1A, 2 and 3). tations in the fnr gene carried by pGS24 (a derivative of pBR322 Activating region 1 (AR1) appears to contact the C-terminal containing the fnr gene in a HindIII-BamHI fragment) were introduced using Taq DNA polymerase and the following synthetic primers, as domain of the RNA polymerase a subunit, thereby preventing described previously (2): 59-GCTTATCATCGATAAGCTTCGTGAATA- inhibition of transcription activation caused by an untethered a 70 TTTTGCCGG (fnr coordinates 1–23) and 59-CGTAGAGGATCCAGGC- subunit. Activating region 3 (AR3) contacts the s subunit of TGTACGC (1625–1641), where the unique HindIII and BamHI targets are underlined. Following digestion with HindIII and BamHI, the polymerase chain reaction products were ligated into the corresponding * This work was supported by the Biotechnology and Biological Sci- sites of pBR322. Plasmids were isolated by standard methods, and ences Research Council of the United Kingdom and the Royal Society. mutations in the fnr gene were defined by Applied Biosystems cycle The costs of publication of this article were defrayed in part by the sequencing with the aid of two primers: 59-AAACATATGGTCCCGGA- payment of page charges. This article must therefore be hereby marked AAAGCG (520 –536) and 59-GGAAACCTCGATGGTAGCTGAAATCCC- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to GTTCG (866 – 898). indicate this fact. Identification of FNR Variants Defective in Repression—The library ‡ To whom correspondence should be addressed. Tel.: 44-114-222- 4403; Fax: 44-114-272-8697; E-mail: [email protected]. of mutagenized fnr genes in pBR322 was used to transform JRG3701, a 10244 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. FNR-mediated Repression 10245 derivative of JRG1728 [D(lacIPOZYA)X74 galU galK rpsL D(ara-leu) were produced in normal amounts, as judged by Western blot- D(tyrR-fnr-rac-trg)17 zdd-230::Tn9] containing a compatible yfiD-lac ting (Table I). Expression of fnr is autoregulated, partially by reporter plasmid, pGS1000 (10). Transformants were tested for en- binding at a site overlapping the transcript start such that hanced yfiD-lacZ expression on L-agar containing: 5-bromo-4-chloro-3- FNR acts as a molecular brick. Hence, the amount of each FNR indoyl b-galactoside (40 mg/ml), ampicillin (200 mg/ml), and tetracycline variant may reflect their relative affinities for this site as well (35 mg/ml). The plates were incubated under anaerobic conditions at 37 °C for 16 h, after which they were exposed to air. The colonies were as any effects that the particular substitutions may have on monitored and those that became blue first were noted and recovered. protein stability. Even those produced at lower levels were The levels of yfiD-lacZ expression were determined by measuring b-ga- expressed well enough for the screening protocol, because, al- lactosidase activity (11) with cultures that had been grown anaerobi- though chromosomal expression yielded least FNR (,5% of cally for 16 h at 37 °C in sealed bottles containing L broth supplemented that obtained with pGS24), it was still sufficient to allow reg- with glucose (0.4% w/v), ampicillin (200 mg/ml), and tetracycline (35 mg/ml). The pBR322 derivatives containing the mutant fnr genes were ulation of yfiD expression comparable with that observed with isolated and were then used to transform JRG3917, a JRG1728 deriv- multicopy fnr (Table I). This reflects the relative abundance of ative containing a pRW2-based ndh-lacZ reporter, pndh (2), and the the reporter plasmid (2–5 copies per cell) compared with the degree of repression conferred by the FNR variants was estimated by fnr-encoding plasmid (15–20 copies per cell). Therefore, the measuring b-galactosidase activity as above. As anaerobic ndh-lacZ failure to down-regulate yfiD is probably not due to a lack of expression of fnr strains is enhanced during growth on rich medium (6), FNR in the cell. the effect of the FNR variants on ndh-lacZ expression was also deter- Nucleotide sequence analysis revealed that most of the FNR mined following growth on a glucose minimal medium supplemented with leucine (12) and appropriate antibiotics. Similarly, two simple variants encoded by the plasmids contained substitutions in FNR-activated promoters, the Class I FF-71.5pmelR (FNR site at the face of FNR that contains AR1 (Table I, Fig. 1A). The only 271.5) and Class II FFpmelR (FNR site at 241.5), as well as pyfiD exception was the variant M147T (which was isolated twice) promoter mutants with impaired FNR sites at either 293.5 (pyfiD2/1; representing a replacement buried in the dimer interface (helix pGS1062) or 240.5 (pyfiD1/2; pGS1063) fused to lac in pRW50 were a ). There is no obvious reason why such a substitution should used to determine the effects of the selected amino acid substitutions on FNR-mediated activation (9, 13). Western blotting of the soluble frac- result in the properties observed; however, it should be noted tion of sonic cell-free extracts with polyclonal anti-FNR serum has been that the M147T variant still retained significant repressing described previously (14). The relative amount of each FNR variant was ability (Table I). Many of the substitutions (K54E, Y69C, R72L, estimated by quantitative densitometry using a Vilber-Lourmat imag- G74C, F92S, A95P) were clustered in a series of loops that form ing system. the AR1 side of the FNR b-roll. A second cluster was evident in Partial Proteolysis—The FNR protein was purified from a glutathi- the region encompassing a to a (S178F, F191L, L193P, one S-transferase-FNR fusion protein as described (15). Aliquots (20 ml, D E 3.4 mg/ml) of the isolated FNR were incubated at 30 °C for up to2hin R197H). Four variants (G74C, F92S, A95P, and L193P) had the presence of either 0.68 unit of trypsin (Sigma), 1 unit of chymotryp- been identified in a previous screen, and it was suggested that sin (Sigma), or 0.1 unit of V8 protease (Sigma). The peptides generated the defect in yfiD down-regulation was due to an altered AR1- were then fractionated by SDS-polyacrylamide gel electrophoresis and containing surface in these variants (9). Five of the 17 variants transferred to nitrocellulose membranes for N-terminal amino acid identified contained two substitutions, but in each case at least sequencing. one of the replacements was of a residue predicted to be close to RESULTS or part of the AR1-containing surface. The pBR322 derivatives encoding FNR variants defective in Identification of Repression Defective FNR Variants—Previ- yfiD down-regulation were transferred into JRG3917 (Dfnr ous attempts to isolate repression defective FNR variants using Dlac, containing a compatible FNR-repressed ndh-lacZ re- the FNR-repressible ndh promoter as a screen were unsuccess- porter, pndh; Ref. 2). All the variants failed to repress ndh ful, yielding only FNR proteins with reduced affinity for their expression normally indicating that the yfiD and ndh promot- DNA target and therefore compromised in both activation and ers may share a common mechanism of FNR-mediated repres- repression (16). However, recent analysis of the regulation of sion (Table I). As anaerobic expression of ndh is known to yfiD expression in E. coli (9) has provided an opportunity to respond to nutrient quality in fnr strains (6), ndh-lacZ expres- develop a better screen. The yfiD gene has an unusual pro- sion was also determined for cultures grown in a defined min- moter architecture for an FNR-activated gene, with two FNR imal medium (Table I). The data obtained confirmed the FNR sites centered at 240.5 and 293.5 relative to the transcript repression defects observed in rich medium. Tests with equiv- start. Multiple FNR sites are usually found in FNR-repressed alent strains carrying the FNR-activated Class II FFpmelR- promoters; for example, the ndh gene has FNR sites centered lacZ reporter plasmid, which should be substantially unaf- at 250.5 and 294.5 (1). Expression of yfiD is dependent upon fected by substitutions in AR1, indicated that all but four of the an activating FNR dimer centered at 240.5, but occupation of seventeen variants were capable of activating transcription. It the upstream FNR site (293.5) down-regulates expression (9). was expected that all the variants would be competent activa- Therefore, screening a library of randomly mutagenized fnr tors of Class II promoters, because the basis of the original genes for those that allow increased yfiD-lacZ expression (i.e. screen depended on FNR activation from a site at 240.5 in the FNR variants that still activate from position 240.5, but fail to yfiD promoter. Therefore, the response of the simple Class II act as repressors at 293.5) should ensure that the FNR pro- promoter FFpmelR defines two types of FNR variant: Type A, teins selected are not compromised in DNA binding activity. Error-prone polymerase chain reaction mutagenesis was which fails to repress but activates normally, and Type B, which has both repression and activation defects. This assign- used to generate a library of randomly mutated fnr genes in ment was confirmed using a pyfiD2/1 reporter (pGS1062) with pBR322. Transformants of JRG3701 (Dfnr Dlac, contain- ing a compatible yfiD-lacZ reporter plasmid, pGS1000) were an impaired FNR site at 293.5 (Table I). The Type B variants exhibited much reduced activity at this promoter compared screened for elevated yfiD-lacZ expression on 5-bromo-4- chloro-3-indoyl b-galactoside plates. Approximately 4000 colo- with the Type A proteins. Studies with the Class I promoter a contact for activation, nies were screened, and 19 were found to contain plasmids that FF-71.5pmelR, which requires an AR1- enhanced yfiD expression (Table I). Estimation of the b-galac- indicated that the Type A substitutions could be neutral for tosidase activities of anaerobic cultures indicated that all of the (R197H), or improve (K54E, A95P) or impair (I45T, Y69C, FNR variants encoded by the plasmids were defective in the S133P, G74C, M147T, L193P, L239P, L239E), the AR1 contact. down-regulation of yfiD expression. Most of the FNR variants However, any improvement in the AR1 contact was insufficient 10246 FNR-mediated Repression TABLE I Transcription regulation of FNR-repressed and FNR-activated promoters by 17 FNR variants Expression of b-galactosidase driven by the indicated FNR variants was measured in JRG1728 (Dlac Dfnr) transformed with pyfiD,pndh, FFpmelR, or FF-71.5pmelR fused to lac. Values (which varied by no more than 10%) are the mean of duplicate assays of at least two independent anaerobic cultures of each strain, grown on rich medium in sealed bottles at 37 °C for 16 h. Expression of ndh-lacZ was also determined in minimal medium to eliminate the enhancement of ndh expression associated with anaerobic growth on rich medium in the absence of FNR. Aerobic expression from the test promoters for all the variants was similar to that observed for FNR: 100 Miller units for pyfiD; 2100 for pndh; 110 for pyfiD2/1; 150 for FFpmelR; and 150 for FF-71.5pmelR. Expression of each variant was assessed by Western blotting using anti-FNR serum and is given as a percentage (620%) of that observed with cultures expressing fnr from an equivalent plasmid (pGS24, row labeled FNR). The figures in parentheses indicate the number of independent isolates. Anaerobic b-galactosidase activity Codon FNR variant Expression change pndh defined pyfiD pndh pyfiD2/1 FFpmelR FF-71.5pmelR medium % Miller units Dfnr 70 4600 2400 110 70 140 fnr ,5 760 420 6300 2600 FNR 100 680 170 510 8220 6220 3160 Type A: S13P TCT-CCT 100 6750 4790 2300 6710 2440 1090 S145N AGC-AAC I45T ATC-ACC 100 1430 340 800 7180 5790 2830 K54E AAA-GAA 100 3440 510 1120 8160 9790 4580 Y69C TAT-TGT 100 5620 1040 1470 7020 3600 1640 S133P TCC-CCC G74C GGT-TGT 30 5300 4000 1400 7240 5000 650 A95P GCA-CCA 100 2200 1000 1800 5070 7700 4100 M147T (2) ATG-ACG 100 1840 1120 1760 7880 4620 2510 K164E AAA-GAA 100 6750 2710 1840 7450 2730 1000 F191L (2) TTC-CTC L193P CTG-CCG 50 4600 3000 1800 7975 7600 1770 R197H CGT-CAT 100 2100 1130 1660 8110 7160 3020 L239P CTG-CCG 30 4710 1390 1820 9240 4290 1960 L239E CTG-CAG 20 4570 1080 2400 8595 5140 2140 Stop-14 C-terminal amino acid TGA-CGA 20 6470 1680 1250 9450 3680 1670 Type B: L34P CTC-CCC 30 3590 4720 1290 3630 340 290 R72L CGC-CTC 5 6440 4350 1540 5250 880 570 L193P CTG-CCG F92S TTC-TCC 20 5300 6710 2500 1740 285 290 S178F TCC-TTC 100 5640 3900 1990 5910 1110 490 fnr expressed from the chromosome. This variant also contained the substitution C16S, which is known to be neutral for FNR activity. The additional 14 C-terminal amino acids are: RFFRITHYPFCHNH. FIG.1. Positions of amino acid replacements that compromise FNR-mediated repression and locations of solvent exposed regions of FNR. A, predicted structure of an FNR monomer based on that of the cAMP receptor protein showing the positions of amino acid replacements that impair FNR-mediated repression of ndh-lacZ and yfiD-lacZ expression. The helix-turn-helix motif (a -a ) in the DNA-binding domain, the D F essential cysteine residues that act as ligands for the [4Fe,4S] cluster (ringed), and the previously identified activating regions AR1 and AR3 (or 85-loop) are also indicated. B, surface-exposed regions of FNR. Predicted structure of an FNR monomer showing the sites of cleavage by trypsin and V8 protease. to allow activation from a yfiD reporter with an impaired FNR Substitutions Effecting FNR-mediated Repression Are Lo- site at 240.5, pyfiD2/1 (not shown). Furthermore, the variants cated in Surface-exposed Regions—There is no structure avail- G74C and L193P, which activated the Class II promoter well, able for FNR, but there is evidence to indicate that the pre- but were poor at Class I, indicated that the anti-inhibition dicted similarity with the cAMP receptor protein is well contact made between the RNA polymerase a subunit and the founded (1). An essential feature of any regions of FNR in- AR1 face of FNR (required at Class II promoters) is different to volved in contacting other components of the transcription the activating AR1 a subunit contact made at Class I machinery is that they must be solvent exposed. Partial prote- promoters. olysis of isolated FNR with trypsin (28 possible cleavage sites, FNR-mediated Repression 10247 TABLE II Partial proteolysis of FNR Polypeptide products identified following digestion of FNR (68 mg) with trypsin (a) or V8 protease (b). The FNR protein used was FNR-572 released from a glutathione S-transferase-FNR fusion protein (15) as a result, the isolated FNR protein contains an additional 15 N-terminal amino acids (GSPGISGGGGGILDS). Lowercase letters indicate that the amino acid residue was not detected but is from the known se- quence; – indicates that the sequence corresponds to the N-terminal of FNR; the apparent molecular weights (M ) are estimated from compar- isons with standard proteins on the blots. Band and apparent M N-terminal sequences Cleavage position (a) 1 37,000 GSPGIS – 2 36,000 gsPGIsGG – 3 32,000 GSPGISGG – 4 28,000 GSPGISGG – SYTITEQG Lys tITEQG Tyr 5 25,000 SYTITEQGDEQITGF Lys TITEQGDEQITgFHL Tyr 6 22,500 SYTITEQGDEQITGF Lys TITEQGDEQITGF Tyr 7 21,000 HLAGdLVG Phe SYTITEQG Lys TITEQGdE Tyr FIG.2. Positions of amino acid substitutions in FNR defining 8 17,000 SYTITEQGDEQITGF Lys activating regions and repression defective variants superim- TITEEQGDEQITGFHL Tyr posed on the a backbone of a cAMP receptor protein monomer. 9 14,500 GSPGISGGGGGILDS – The position of amino acids forming AR1 (see Refs. 3 and 18) important 72 73 118 120 SYTITEQGDEQITGF Lys in transcription at Class I promoters (Arg , Ser , Thr , Met , 181 184 186 187 188 225 TITEQGdEQITGFHL Tyr Phe , Arg , Phe , Ser , Pro , and Ala ; blue), AR3 (19) re- 81 85 86 10 13,000 GSPGISGGGGGILDS – quired for the activation of Class II promoters (Ile , Gly , Asp , and 112 45 54 69 SYTITEQGDEQITgFH Lys Phe ; green); and repression-defective variants (Ile , Lys , Tyr , 74 92 95 178 193 197 TITEQGDEQITgFH Tyr Gly , Phe , Ala , Ser , Leu , and Arg ; red) are indicated. The 72 191 IIRRiQsggcAiH Arg yellow spheres indicate positions (Arg and Phe ) that are common to GFSPREFRLTMTR Arg AR1 activation and repression. The AR3 defining residues of FNR are (b) clustered on the left (green); the AR1 defining amino acids (blue and 1 33,000 KRIIRRI Glu yellow) form a large surface along the opposite face of FNR to that 2 28,000 LDQLDNIIERKKPIQ Glu containing AR3. The repression-defective mutations encode substitu- 3 17,000 KRIIRRIOsG Glu tions along the AR1 surface (red and yellow residues) and a further TSMVcEIPFE Glu region falling between AR3 and AR1 (red residues only) forming a IPFETLDDS Glu sharks fin-like pattern of substitutions. The repression defective vari- 13 34 239 4 15,500 KRIIRRIQSGG Glu ants with substitutions at Ser , Leu , and Leu lie in regions of FNR TSMVcEIPFETL Glu beyond that encompassed by the cAMP receptor protein structure, 147 133 145 164 Met in the dimer interface, and the Ser , Ser , and Lys (iso- lated as double mutations with partners in the AR1 face) are omitted for 406 possible peptides) or V8 protease (28 possible cleavage clarity. sites, 435 possible peptides) followed by N-terminal amino acid sequencing of the resulting peptides revealed that the substi- direct contact with RNA polymerase to inhibit transcription tutions affecting repression were within regions of FNR acces- initiation (17). The observation that FNR and RNA polymerase sible to the proteases (Table II, Fig. 1B). Following digestion can simultaneously interact with the FNR-repressible ndh pro- with trypsin, separation of peptides by SDS-polyacrylamide gel moter suggested that, in this case, repression is unlikely to be electrophoresis, and blotting, 10 Coomassie Blue-stained bands mediated simply by promoter occlusion (6, 7). The ndh pro- were detected. The N-terminal amino acid sequences of the moter has two FNR sites centered at 250.5 and 294.5, and it peptides present in each band were determined (Table IIa). has been proposed that FNR occupation of these sites prevents Many of the bands contained the same N-terminal sequences, RNA polymerase a subunit from interacting with DNA (7). indicating preferential cleavage at a limited number of posi- Such a repression mechanism may require direct protein con- tions with subsequent C-terminal processing. The preferred 6 77 79 tacts between the two FNR dimers and/or FNR and RNA po- tryptic/chymotryptic cleavage points were: Arg , Lys , Tyr , 92 184 lymerase. Previous attempts to identify FNR variants compro- Phe , and Arg (Fig. 1B). Treatment with V8 protease pro- mised for repression of ndh-lacZ were unsuccessful, because duced four bands with the N-terminal sequences in Table IIb. 4 38 117 the transformants recovered contained plasmids encoding FNR These data imply cleavage at positions Glu , Glu , Glu , and proteins with defects in DNA binding (16). However the yfiD- Glu . Therefore, the region encompassing the series of loops lacZ reporter provided an opportunity to screen out FNR vari- in the AR1 side of the b-roll was particularly susceptible to ants defective in DNA-binding, because this promoter, al- proteolytic cleavage. These results indicate that the regions though down-regulated by FNR occupation of a site centered at identified as important for FNR-mediated repression are sol- 293.5, requires an activating FNR dimer (at 240.5) for vent exposed and also provide further evidence to support the expression. cAMP receptor protein-based predicted FNR structure, because Using yfiD-lacZ as an initial screen 17 FNR variants defec- the equivalent positions are known to be surface exposed in the tive in repressing both yfiD and ndh promoters were identified. cAMP receptor protein. All but one of the variants (M147T) had substitutions near or DISCUSSION within the face of FNR that contains AR1 (Fig. 1, 2), a region of Transcription can be repressed either passively by promoter the protein known to contact the a subunit of RNA polymerase occlusion, i.e. when a regulator blocks access of RNA polymer- and thereby facilitating transcription activation (3). Two types ase to the promoter, or actively, in which the regulator makes of contact can be made depending on the architecture of the 10248 FNR-mediated Repression activated promoter. At Class I promoters (FNR site at or be- domain of the a subunit of RNA polymerase thought to be yond 261) AR1 of the downstream FNR monomer makes an involved in GalR-mediated activation also relieve GalR-medi- activating contact with the a subunit, whereas at Class II ated repression (20). However, the activator complex at galP2 promoters (FNR site at or about 241) AR1 makes an anti- is an open complex, whereas the repressing complex at galP1 is inhibition contact (3). It is now apparent that the same face of a closed complex and thus the context of the GalR-RNA polym- FNR can be involved in repressing transcription at promoters erase contacts are different. These context effects are proposed that contain multiple FNR sites and that the regions involved to be sufficient to allow a single regulatory protein to act as a are solvent exposed. This is supported by the observations that both a repressor and an activator while maintaining similar 72 191 (i) the positions of two substitutions (Arg and Phe , indi- regulator-polymerase contacts (20). Therefore, it is suggested cated in yellow on Fig. 2) are common to FNR variants with that the context in which FNR finds itself at the yfiD and ndh altered AR1 or repression properties; (ii) all the repression promoters favors the formation of a ternary complex incorpo- defective variants (with the exception of the M147T) contain a rating FNR-polymerase contacts that render the complex in- substitution in the AR1 side of the FNR monomer that forms competent for transcription activation. two clusters overlapping the previously defined positions of In conclusion, the FNR variants identified here provide the AR1 (Fig. 2); (iii) most of the repression defective variants first indication that specific regions (amino acids) of FNR that display altered activation from a model Class I (AR1-depend- overlap AR1 may be required for transcription repression at ent) promoter (Table I); and (iv) that several amino acids close promoters with multiple FNR sites. Mutational analysis of the to those substituted in repression defective variants (Leu , C-terminal domain of the RNA polymerase a subunit should 74 92 178 Gly , Phe , Ser ) are sufficiently exposed to allow proteo- determine whether FNR can repress transcription via direct 38 77 92 184 lytic attack (Glu , Lys , Phe , and Arg ). Thus, the sim- contact between the AR1 containing face and RNA polymerase plest explanation for the repression defective phenotype dis- or whether FNR-FNR contacts are the key to FNR-mediated played by the FNR variants is that they possess an altered repression at promoters sharing the ndh architecture. surface that is different from but overlaps AR1 which has a role Acknowledgments—We thank J. R. Guest, S. J. W. Busby, and H. J. in repression of promoters with multiple FNR sites. Wing for many helpful discussions and J. Keen for amino acid It has been suggested recently that transcription regulators sequencing. can be viewed as catalysts (20). 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Published: Apr 1, 1999

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