GcvA interacts with both the a and r subunits of RNA polymerase to activate the Escherichia coli gcvB gene and the gcvTHP operon

The glycine cleavage enzyme system in Escherichia coli provides one-carbon units for cellular methylation reactions. The gcvB gene encodes two small RNAs that in turn regulate other genes. The GcvA protein is required for expression of both the gcvTHP (P gcvT ) and gcvB (P gcvB ) promoters. However, the architectures of the two promoters are diﬀerent, with the P gcvT promoter representing a class III activator-dependent promoter and the P gcvB promoter representing a class II activator-dependent promoter. The RNA polymerase holoenzyme was examined for its role in transcription activation of the gcvTHP operon and the gcvB gene by the GcvA protein. The results suggest that GcvA interacts with the RNA polymerase a subunit for activation of the gcvTHP operon and interacts with the RNA polymerase r subunit for activation of the gcvB gene. (cid:1) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.


Introduction
In Escherichia coli the glycine cleavage (GCV) enzyme system, encoded by the gcvTHP operon and lpd gene, catalyzes the cleavage of glycine into CO 2 + NH 3 and transfers a one-carbon unit (C1) to tetrahydrofolate, producing 5,10-methylenetetrahydrofolate [1]. The gcvB gene encodes two small RNAs that, in turn, regulate other pathways (for example, oppA and dppA, encoding the oligopeptide and dipeptide periplasmic binding proteins, respectively) [2]. Both gcvTHP and gcvB are activated by the GcvA protein in response to glycine and repressed by the GcvA + GcvR proteins in the absence of glycine; this repression is enhanced in the presence of purines [2,3]. GcvRÕs ability to repress is dependent on GcvA [4] and genetic and biochemical evidence suggests that GcvR interacts directly with GcvA rather than binding to DNA to cause repression [5,6].
At many promoters, transcription activation occurs through the formation of specific contacts between a DNA-bound activator protein and RNA polymerase (RNAP) [7]. At class I promoters, activators bind upstream of the RNAP binding site [8]. At class II promoters, activators bind to sites that are adjacent to or overlap the RNAP binding sites [9,10]. At class III promoters, multiple activator molecules typically bind more than 90 bps upstream of the RNAP binding site [7,11].
The GcvA protein binds to 3 sites in the gcvTHP control region, from bp À271 to À242 (site 3), from bp À241 to À214 (site 2) and from bp À69 to À34 (site 1) relative to the transcription initiation site ( Fig. 1(a)) [12]. All three binding sites are required for 0378 GcvA AE GcvR-mediated repression of the gcvTHP operon, whereas only sites 3 and 2 appear to be required for GcvA-mediated activation of the operon in response to glycine [12]. Lrp, a global regulator for many genes involved in amino acid metabolism [13], binds to multiple sites upstream of the P gcvT promoter, from bp À92 to À229 ( Fig. 1(a)) and plays a structural role in gcvTHP regulation, bending the DNA to allow GcvA to function as either an activator or repressor [14]. The cAMP receptor protein (CRP) binds to a site from bp À299 to À326 relative to the transcription initiation site ( Fig. 1(a)) and is required for a 4-fold activation of the gcvTHP operon in glucose minimal (GM) medium [15]. Thus, P gcvT is a class III promoter. The gcvB promoter is divergently transcribed from and overlaps the gcvA promoter [2,12]. GcvA binds to a single region in the gcvB control region from bp À29 to À76 relative to the transcription initiation site ( Fig. 1(b)) [12], and binding of GcvA to this region is required for both GcvA-mediated activation in the presence of glycine and GcvA AE GcvR-mediated repression in the absence of glycine [16]. Thus, P gcvB is a class II promoter.
Because of the different architectures of the P gcvT and P gcvB promoters, we tested if GcvA interacts with differ-ent subunits of RNAP at these two promoters. Our results are consistent with GcvA interacting with the RNAP a subunit to activate P gcvT and interacting with the RNAP r subunit to activate P gcvB .

Strains and plasmids
The E. coli strains and plasmids used in this study are listed in Table 1. Strain CAG20207 carries the X(Cm R )ptrp-rpoD fusion and was provided by Dr. C. Gross. Plasmids encoding wild-type (WT) r 70 and alanine substitutions in the r 70 CTD in residues 590 through 613 [9] are variants of pGEX-2T in which the coding region of glutathionine S-transferase is fused to RpoD at amino acid 8 and expressed from the tac promoter and were provided by Dr. C. Gross.

Phage
The kgcvT::lacZ translational fusion phage includes 466 bp upstream and 291 bp downstream of the P gcvT Low-copy plasmid carrying WT gcvA [29] a All strains except CAG20207 also carry D(argF-lac)U169, pheA905, thi, araD129, rpsL150, relA1, deoC1, flb5301, ptsF25 and rpsR mutations.  Fig. 1. The E. coli P gcvT and P gcvB promoter regions. (a) The E. coli P gcvT promoter. The transcription initiation site (+1), the promoter À10 and À35 regions [17], and the GcvA [12], Lrp [3], PurR [30] and CRP [15] binding sites are indicated. (b) The E. coli P gcvB promoter. The gcvB transcription initiation site (+1) and the promoter À10 and À35 regions [2] are indicated below the sequence. The gcvA transcription start site (+1) and the promoter À10 and À35 regions [12,16] are indicated above the sequence. The GcvA binding site [12] is indicated above the sequence. The sites are approximately to scale. transcription initiation site fused in phase with the lac-ZYA genes [17]. The kgcvB::lacZ transcriptional fusion phage includes 150 bp upstream of the P gcvB transcription initiation site fused at bp +50 within gcvB to translationally competent lacZYA genes [2]. The kgcvA::lacZ translational fusion phage includes 231 bp upstream and 218 bp downstream of the gcvA transcription initiation site fused in phase with the lacZYA genes [18]. The kgcvR::lacZ translational fusion phage includes 348 bp upstream and 132 bp downstream of the gcvR transcription initiation site fused in phase with the lacZYA genes [19]. Each fusion carries all of the known regulatory sites for the respective promoters. These phage were used to lysogenize various host strains and each lysogen was tested to ensure that it carried a single-copy of the k chromosome by infection with kcI90c17 [20]. Lysogens were grown at 30°C since all of the fusion phage carry the kcI857 mutation, resulting in a temperature sensitive repressor. Strains carrying the X(Cm R )ptrp-rpoD allele were difficult to lysogenize directly with the k fusion phage. Therefore, single-copy lysogens were first constructed in strain GS162 and the X(Cm R )ptrp-rpoD allele introduced into the lysogens by P1clr transduction as described [21] and are designated as GS1142 derivatives.

Media
The complex medium used was Luria-Bertani broth (LB) [21]. The GM medium used was the salts of Vogel and Bonner [22] supplemented with 0.4% glucose. Supplements were added at the following concentrations in lg ml À1 : glycine, 300; phenylalanine, 50; inosine, 50; thiamine, 1. GM medium was always supplemented with phenylalanine and thiamine since all strains carry the pheA905 and thi mutations.

b-Galactosidase assays
Cells were grown in the media indicated in the text to an OD 600 of $0.5, placed on ice for 20 min and b-galactosidase levels determined using the chloroform-SDS lysis procedure [21]. All assays were performed at least twice and the activity of each sample was determined in triplicate.

Effects of deletions of the aCTD on P gcvT and P gcvB expression
The P gcvT promoter shows characteristics of a class III promoter, whereas the P gcvB promoter shows characteristics of a class II promoter ( Fig. 1(a) and (b)). Despite the different architectures of the two promoters, a region of GcvA defined by the gcvA -F31L mutation in the helix-turn-helix (H-T-H) region is required for GcvA-mediated activation of both P gcvT and P gcvB [2,23]. Since GcvA contacts the aCTD to activate P gcvT [24], we tested if the aCTD is also required for activation of P gcvB . The lysogens GS1053kgcvT::lacZ and GS1053kgcvB::lacZ were transformed with plasmid pREIIa, encoding a WT RNAP a subunit, and plasmids pGS490 and pGS491, encoding deletions of the aCTD from amino acid residue 239 (RpoAD239) and 245 (RpoAD245), respectively. We used the gcvR mutant strain GS1053 for these studies to insure that any decrease in gcvT::lacZ or gcvB::lacZ repression could not be attributed to the GcvA AE GcvR repression system. The transformed cells were grown in LB + ampicillin (Ap) to mid-log phase of growth and assayed for bgalactosidase activity. As reported previously, GS1053kgcvT::lacZ transformed with plasmids pGS490 and pGS491 displayed about 2-fold lower b-galactosidase levels compared to the transformant encoding the WT a subunit (Fig. 2(a)). Although the effects of the rpoA mutations were not dramatic on GcvA activation of the gcvT::lacZ fusion, this is not unexpected as similar results have been reported in other studies for single amino acid changes and deletions of the aCTD [25]. GS1053kgcvB::lacZ transformed with plasmids pGS490 and pGS491 displayed b-galactosidase levels essentially the same as the transformant encoding the WT a subunit ( Fig. 2(b)). These results suggest that the aCTD is required for activation of gcvT::lacZ, but is not required for activation of gcvB::lacZ.

Effects of changes in the r 70 CTD on P gcvT and P gcvB expression
Since the aCTD is required for GcvA-mediated activation of P gcvT , but not for GcvA-mediated activation of P gcvB , it was of interest to examine whether the RNAP r 70 subunit is required for activation of either P gcvB or P gcvT . The X(Cm R )ptrp-rpoD fusion was introduced into lysogens GS162kgcvT::lacZ or GS162k gcvB::lacZ by P1clr transduction, generating lysogens GS1142kgcvT::lacZ and GS1142kgcvB::lacZ, respectively. In these lysogens, the chromosomal rpoD gene is under the control of the trp promoter to minimize expression of endogenous r 70 [9]. These strains were transformed with pGEX-2Tr 70 encoding the WT r 70 and with pGEX-2T derivative plasmids encoding alanine substitutions in the r 70 CTD. Lysogen GS1142kgcvT::lacZ and GS1142kgcvB::lacZ transformed with pGEX-2Tr 70 WT and the alanine substitutions were grown in GM + glycine + Ap + Chloramphenicol (Cm) to midlog phase of growth and assayed for b-galactosidase activity [21]. Lysogen GS1142kgcvB::lacZ transformed with pGEX-2T carrying the alanine substitutions E591A, L595A, H600A, S602A and R603A showed 1.5-to 2-fold reduced levels of gcvB::lacZ expression compared to the transformant carrying WT r 70 (Fig. 3(b)). The remaining substitutions in rpoD had no significant effect on gcvB::lacZ expression. The small effects of the rpoD mutations on GcvA-mediated activation of the gcvB::lacZ fusion are not unexpected, as similar results have been reported in other studies for single amino acid changes in rpoD [9,26]. In vivo assays of the effect of alanine substitutions in r 70 CTD are difficult. The mutant forms of r 70 must compete with the WT r 70 for incorporation into the RNAP holoenzyme. Although the uninduced plasmid encoded pGEX-2Tr 70 WT levels and the levels of the alanine substitutions are equivalent to the expression levels of the chromosomal allele, it is likely that the mutant r 70 proteins with alanine substitutions are unable to compete effectively with the WT r 70 for incorporation into the RNAP holoenzyme [9,26]. Thus, although the rpoD mutations did not have dramatic effects on GcvA-mediated activation in vivo, the results suggest that several residues in the r 70 CTD are likely to be important for GcvA-mediated activation of P gcvB . GS1142kgcvT::lacZ transformed with pGEX-2T carrying alanine substitutions in r 70 displayed b-galactosidase levels essentially the same as the transformant encoding the WT r 70 subunit ( Fig. 3(a)). These results suggest that the rCTD is not required for activation of gcvT::lacZ.
3.3. Reduced gcvB::lacZ expression in the rpoD mutants is not due to increased GcvR levels or decreased GcvA levels Activation of gcvB requires a functional GcvA protein and repression requires a functional GcvR protein [2]. It is possible that the r 70 CTD mutants that reduce gcvB::lacZ expression either decrease the levels of GcvA or increase the levels of GcvR. Either condition would be expected to decrease gcvB::lacZ expression. Although one might expect such changes would also alter gcvT::lacZ expression, it is possible a lower concentration of GcvA is required for activation of the gcvT::lacZ fusion or that a higher concentration of GcvR is required for repression of the gcvT::lacZ fusion. Thus, the X(Cm R )ptrp-rpoD fusion was introduced into lysogens GS162k gcvR::lacZ and GS162kgcvA::lacZ by P1clr transduction, generating lysogens GS1142kgcvR::lacZ and GS1142kgcvA::lacZ. The lysogens were transformed with pGEX-2Tr 70 WT and with pGEX-2T derivative plasmids encoding alanine substitutions in the r 70 CTD (E591A, L595A, H600A, S602A and R603A). The transformants were grown in GM + glycine + Cm + Ap to mid-log phase of growth  I590A  E591A  K593A  L595A  R596A  K597A  L598A  R599A  H600A  S602A  R603A  S604A  E605A  R608A  D612A  D613A  I590A  E591A  K593A  L595A  R596A  K597A  L598A  R599A  H600A  S602A  R603A  S604A  E605A  R608A

Discussion
P gcvT is a class III activator-dependent promoter ( Fig.  1(a)) and P gcvB is a class II activator-dependent promoter ( Fig. 1(b)). Activators for class III promoters generally make a protein AE protein interaction with the aCTD of the RNAP [7,11], whereas activators for class II promoters generally make protein AE protein interactions with the r 70 subunit of the RNAP and often with the a subunit as well [9,10]. Results presented here show that although the RpoAD239 and RpoAD245 deletions of the aCTD result in about 2-fold reduced levels of expression of a gcvT::lacZ fusion, the deletions have no significant affect on expression of a gcvB::lacZ fusion ( Fig. 2(a) and (b)). In addition, a genetic screen of >15,000 PCR mutagenized ropA transformants failed to identify any amino acid in the a subunit necessary for GcvA activation of gcvB::lacZ. Thus, the GcvA AE aCTD interaction required for GcvA activation of the P gcvT promoter is not required for GcvA activation of the P gcvB promoter. The fact that Lrp and CRP are required for full activation of P gcvT [14,15] complicates interpretation of the results. However, evidence suggest that LrpÕs role is structural, bending the P gcvT DNA to allow a GcvA AE RNAP interaction [14] and CRPÕs role is to antagonize GcvA-mediated repression [15]. Thus, although it is possible that part of the effects of the a mutations could be mediated through Lrp and CRP, the results are more consistent with a GcvA AE aCTD interaction likely being required for GcvA-mediated activation at the P gcvT promoter.
Five r 70 CTD mutants, E591A, L595A, H600A, S602A and R603A, were found to be important for PgcvB expression, resulting in 1.5-to 2-fold reduced levels of expression of a gcvB::lacZ fusion (Fig. 3(b)). However, none of the alanine substitutions in RpoD had a significant effect on gcvT::lacZ expression ( Fig. 3(a)). These results suggest that a GcvA AE r 70 interaction is likely required at the P gcvB promoter, but is not required for GcvA activation of the P gcvT promoter. Although decreased GcvA levels or increased GcvR levels could explain the decreased levels of gcvB::lacZ expression, none of the mutations that resulted in decreased gcvB::lacZ expression had a significant effect on either gcvR::lacZ or gcvA::lacZ expression ( Fig. 4(a) and (b)). Thus, the decreases in gcvB::lacZ expression observed are likely due to direct effects of the alanine substitutions in the r 70 subunit preventing necessary GcvA AE r 70 CTD interactions required for activation rather than indirect effects of the mutations altering the levels of the GcvA and GcvR proteins.
Previous genetic results showed that a region of GcvA that is required for activation of P gcvT , defined by the gcvA-F31L positive control mutation, is also required for activation of the P gcvB promoter [2,23]. Modeling studies suggest that the side chain of residue F31 of GcvA is surface exposed, does not interact directly with DNA, and is capable of interacting directly with the aCTD [27]. If the amino acid at position 31 defines a region of GcvA that interacts with RNAP, then the results from this study are consistent with a hypothesis where this region interacts with the RNAP a subunit at a class III promoter (P gcvT ) and interacts with the RNAP r subunit at a class II promoter (P gcvB ). We are constructing and testing  Fig. 4. Effects of alanine substitutions in the rCTD on chromosomal gcvA::lacZ and gcvR::lacZ expression. The X(Cm R ptrp-rpoD) strain GS1142, lysogenized with either a kgcvA::lacZ fusion (a) or a kgcvR::lacZ fusion (b), was transformed with plasmid pGEX-2Tr 70 encoding WT r 70 or alanine substitutions E591A, L595A, H600A, S602A and R603A in the rCTD, cells were grown in GM + glycine + Ap + Cm and assayed for bgalactosidase activity [21]. The activities of the lysogens transformed with plasmids encoding WT r 70 were set at 100%, and the activities of the mutants are given as percentages relative to the WT activity. Activity of GS1142kgcvA::lacZ transformed with a plasmid encoding WT r 70 was 4 ± 0.3 Miller units and the activity of GS1142kgcvR::lacZ transformed with a plasmid encoding WT r 70 was 57 ± 6 Miller units. additional mutations in the GcvA H-T-H region to determine if there are other amino acids in GcvA that are required for activation of both the P gcvT and P gcvB promoters, or are specific for only one of the promoters. Characterization of additional mutations and in vitro transcription assays showing the importance of the RNAP subunits in transcription from the respective promoters will provide results necessary to confirm the above model.