Department of Microbiology, The University of Iowa, Iowa City, IA 52242, USA1
Author for correspondence: George V. Stauffer. Tel: +1 319 335 7791. Fax: +1 319 335 9006. e-mail: george-stauffer{at}uiowa.edu
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ABSTRACT |
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Keywords: gcvTHP, GcvA, GcvR, glycine, repression
Abbreviations: AP, ampicillin; C1, one-carbon; co-ppt, co-precipitation; gcv, gcvTHP operon; GCV, glycine cleavage; GMS, gel mobility shift; LTTR, LysR-type transcriptional regulator; RNAP, RNA polymerase; TPEG, phenylethyl-ß-D-thiogalactoside
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INTRODUCTION |
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The GCV enzyme system consists of four proteins designated T-protein, H-protein, P-protein and L-protein (Kikuchi, 1973 ). The gcvT, gcvH and gcvP genes, encoding the T-, H- and P-proteins, respectively, form an operon at 65·7 min on the E. coli chromosome (Plamann et al., 1983
; Stauffer et al., 1993
, 1994
). The lpd-encoded lipoamide dehydrogenase, common to the pyruvate and 2-oxoglutarate dehydrogenase enzyme complexes, functions as the L-protein (Steiert et al., 1990
). The lpd gene maps at 2·8 min (Berlyn et al., 1996
).
Transcription of the gcv operon is under the control of three global regulatory proteins [Lrp, PurR and cAMP-receptor protein (CRP)] and two gcv-specific transcriptional regulatory proteins (GcvA and GcvR) (Ghrist & Stauffer, 1995 ; Lin et al., 1992
; Stauffer & Stauffer, 1994
; Wilson et al., 1993a
; Wilson & Stauffer, 1994
; Wonderling & Stauffer, 1999
). Together these proteins modulate transcription of the operon in response to the levels of glycine and purines within the cell.
The leucine-responsive protein (Lrp), a global regulator involved in the control of transcription of several genes involved in amino acid metabolism (Calvo & Matthews, 1994 ), binds to multiple sites in the gcv control region from bp -229 to -92 relative to the transcriptional start site (Fig. 1
) (Stauffer & Stauffer, 1994
). Lrps role in gcv regulation appears to be structural, binding and bending the DNA to facilitate the formation of the appropriate regulatory complexes for activation or repression of the operon (Stauffer & Stauffer, 1999
), although the possibility that Lrp also activates transcription through interactions with RNA polymerase (RNAP) or a second regulatory protein has not been completely ruled out.
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CRP binds to a site from approximately bp -303 to -324 relative to the transcription start site (Fig. 1) and is required for a fourfold activation of the gcv operon in glucose minimal (GM) medium (Wonderling & Stauffer, 1999
). The specific mechanism for the observed effect of CRP is unknown. However, the dependence of the CRP effect on the repressor function of GcvA suggests that its role is to antagonize GcvA-mediated repression.
GcvA, a member of the LysR family of transcriptional regulatory proteins, functions as an activator of the gcv operon in the presence of exogenous glycine and as a repressor of the operon in the absence of glycine (Wilson et al., 1993a , b
). GcvA-mediated repression is enhanced by the presence of exogenous purines. GcvA binds to three sites in the gcv control region: site 3 from bp -271 to -242, site 2 from bp -242 to -214 and site 1 from bp -69 to -34 (Fig. 1
). All three of these sites are required for repression of the operon, while only sites 2 and 3 appear to be necessary for activation (Wilson et al., 1995
; Wonderling et al., 2000
). In addition, DNase I footprint studies with GcvA showed hypersensitive cleavage sites in the region between the upstream sites 3+2 and the downstream site 1 when GcvA was bound to these sites. These results suggest that GcvA might also be involved in bending DNA to form appropriate nucleoprotein complexes at the gcv promoter.
GcvR is necessary for repression of the gcv operon (Ghrist & Stauffer, 1995 ). In the absence of GcvR, there is constitutive expression of a gcvT::lacZ fusion, and overproduction of GcvR results in super-repression of the fusion. Recently, we showed that GcvA and GcvR interact in vivo using a LexA-based two-hybrid system (Ghrist et al., 2001
). Since GcvR has no repressor capabilities in the absence of GcvA (Ghrist & Stauffer, 1995
), the results suggest that a GcvR/GcvA interaction might be required for repression. Furthermore, since mutations in any of the three GcvA binding sites result in reduced repression (Wilson et al., 1995
; Wonderling et al., 2000
), at least part of the repression response of the gcv operon likely requires that a GcvR/GcvA repressor complex be bound to gcv DNA.
Most LysR-type transcriptional regulators (LTTRs) are believed to directly bind their specific co-inducer molecules, although in most cases this binding has only been indirectly demonstrated through the isolation of mutants that either no longer respond to their respective co-inducer or have altered co-inducer specificity (Colyer & Kredich, 1996 ; Jørgensen & Dandanell, 1999
; Schell, 1993
). Co-inducer binding by LTTRs is manifested in several ways including altered DNA binding and DNA bending, which facilitate LTTR activation or repression of transcription (Gao & Gussin, 1991
; Wek & Hatfield, 1988
; van Keulen et al., 1998
; Ogawa et al., 1999
; Hryniewicz & Kredich, 1994
; Wang et al., 1992
). How glycine and purines affect GcvA and GcvR to allow an appropriate response at the gcv promoter is unknown. In the present study, we determined the likely role glycine plays as a co-inducer in transcriptional control of the gcv operon. The results suggest that GcvA/GcvR-mediated regulation of gcv utilizes a mechanism that may be unique among LTTR regulated operons.
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METHODS |
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Protein purification.
Plasmid pGS473 carries gcvA on an EcoRIHindIII fragment (Jourdan & Stauffer, 1999 ). In this construct gcvA is under transcriptional control of the inducible lac promoter, contains an artificial ShineDalgarno sequence and has six histidine codons at the 3' end of the gcvA ORF. pGS473, however, did not express the hexa-histidine-tagged GcvA (GcvA6xhis) to levels sufficient for use in this work. To increase levels of GcvA6xhis we constructed pGS498 as follows. pGS473 was digested with EcoRI, the ends filled using the large fragment of DNA polymerase I and XbaI linkers added. The resulting fragment was then digested with XbaI/HindIII, the fragment gel-purified and ligated into the corresponding sites in the pET-3d vector (New England Biolabs), placing the gcvA6xhis allele under transcriptional control of the T7 promoter. Since GcvA and GcvR interact in vivo (Ghrist et al., 2001
), pGS498 was used to transform the
gcvR::KNr strain GS1128 on LB agar+AP, and an APr transformant isolated. Expression of GcvA6xhis in strain GS1128 prevents the possible co-purification of GcvA6xhis and WT GcvR. The APr transformant was used to inoculate 3 ml Terrific Broth+AP and the culture was incubated at 37 °C for 3 h. To prevent the accumulation of ß-lactamase in the growth medium that could result in the loss of the expression plasmid from cells and lower protein expression, cells from the 3 ml culture were harvested by centrifugation and used to inoculate 1 l Terrific Broth+AP. The culture was grown at 30 °C to OD600 between 1·2 and 1·4. Expression of the T7 RNAP was induced by the addition of IPTG, resulting in transcription of gcvA6xhis. Induced cultures were then incubated at 24 °C for 7 h, the cells harvested by centrifugation, resuspended in loading buffer A (50 mM HEPES/NaOH pH 7·4, 500 mM NaCl, 1 mM EDTA, 0·1% Triton X-100) and sonicated. The lysate was clarified by centrifuging at 12000 r.p.m. in a Sorvall SS-34 rotor for 30 min at 4 °C, followed by centrifugation at 40000 r.p.m. in a Beckman 60Ti rotor for 1 h at 4 °C. Solid ammonium sulfate was added to 33% saturation and incubated further at 4 °C for 2 h. The precipitate was recovered by centrifugation, dissolved in 5 ml loading buffer A and loaded onto a 5 ml Hi Trap metal chelating column (Pharmacia Biotech) pre-equilibrated with loading buffer A. The column was washed with 20 vols loading buffer A followed by 10 column vols wash buffer A (50 mM HEPES/NaOH pH 7·4, 500 mM NaCl, 0·1 mM EDTA, 10% glycerol, 20 mM imidazole). GcvA6xhis protein was then eluted from the column with a 20500 mM gradient of imidazole in wash buffer A. GcvA6xhis eluted from the column at approximately 125 mM imidazole. Fractions containing GcvA were pooled, concentrated using an Amicon membrane ultrafiltration system with a 10000 molecular mass cut off and stored at 4 °C in wash buffer A+125 mM imidazole.
The gcvR ORF was cloned into the vector pTBY1 of the Impact I protein purification system (New England Biolabs) as follows. The gcvR ORF carried on pBR322 was PCR amplified using a 5' primer that encoded an XbaI restriction site, the ShineDalgarno sequence from the E. coli rpoA gene and the first six codons of the gcvR ORF, and a 3' primer that encoded the last six codons of the gcvR ORF in-frame with the first six codons of the intein autocatalytic cleavage signal (which contains a natural KpnI restriction site). The PCR product and pTBY1 vector were digested with XbaI/KpnI, the appropriate DNA fragments were purified from a 1% agarose gel, and ligated. The products of the ligation were used to transform the gcvA::SPr strain GS1145 to prevent co-purification of GcvR and WT GcvA, and transformants selected on LB agar+AP. One APr transformant was single-colony purified and grown in LB+AP, plasmid DNA was isolated and the presence of the PCR product confirmed by digestion with XbaI/KpnI. DNA sequencing of the plasmid using the T7 forward and intein reverse primers (New England Biolabs) confirmed that the GcvRintein reading frame was maintained. This plasmid, designated pGcvR-Impact, was the parent vector used to construct mutant gcvR expression vectors as follows. Mutant gcvR alleles were PCR amplified as described above and cloned into the XbaI/KpnI restriction sites of pGcvR-Impact.
Growth of strain GS1145 carrying pGcvR-Impact (or mutated gcvR alleles), induction of the GcvR::intein::chitin binding domain fusion, and preparation of the cleared lysate were performed as described above for GcvA6xhis except that loading buffer B contained 20 mM HEPES/NaOH pH 7·4, 500 mM NaCl, 1 mM EDTA, 0·1% Triton X-100. The cleared lysate was loaded at a rate of <0·5 ml min-1 onto a Chitin affinity matrix column (New England Biolabs) pre-equilibrated with loading buffer B, washed with 25 column vols loading buffer B followed by 10 column vols wash/elution buffer (20 mM HEPES, pH 7·4, 150 mM NaCl, 0·1 mM EDTA, 10% glycerol). The column was then washed with 3 column vols wash/elution buffer containing 30 mM DTT, and the column left static overnight at 4 °C to allow cleavage of the intein moiety of the fusion. Native GcvR protein was eluted from the column with wash/elution buffer and stored at 4 °C.
Gel mobility shift (GMS) and DNA bending assays.
GMS assays were performed as described (Fried & Crothers, 1981 ; Garner & Revzin, 1981
). DNA fragments were prepared as follows. Plasmid pGS318 carries an in-frame gcvT::lacZ fusion (Stauffer & Stauffer, 1998a
). Plasmids pGS420 and pGS428 were derived from pGS318 and carry a 15 bp and a 25 bp insert between GcvA binding site 3+2 and site 1, respectively (Stauffer & Stauffer, 1998b
). Plasmid pGS357 has a 4 bp change in site 1 that reduces GcvA binding to site 1 about twofold (Wilson et al., 1995
; Wonderling et al., 2000
). These plasmids were used as the starting templates in PCR reactions with primers containing XbaI sites and sequences complementary to gcv upstream and downstream the GcvA binding sites. The resulting 244, 259 and 264 bp DNA fragments generated from pGS318, pGS420 and pGS428, respectively, carry GcvA binding sites 3+2+1, and were cloned into the XbaI site of plasmid pBend2. Five different restriction enzymes were then used to generate DNA fragments of identical length (365 bp), but with different locations of the GcvA binding sites (Fig. 2
). The fragments were 32P-labelled at their 5' termini with T4 polynucleotide kinase (Sambrook et al., 1989
). Target DNA was added to 20 µl reaction mixtures containing DNA binding buffer (10 mM Tris/HCl pH 7·5, 50 mM KCl, 0·5 mM EDTA, 5% glycerol, 1 mM DTT) plus 125 µg BSA ml-1. Reaction mixtures were pre-incubated for 5 min at 37 °C, 2 µl purified GcvA6xhis was added at the concentrations indicated in Figs 3
and 5, and incubation continued for 15 min. One microlitre of loading dye (0·1% xylene cyanol and 50% glycerol in water) was added to each reaction mixture and the samples were loaded onto a 5% polyacrylamide gel and run at 12 V cm-1. Gels were transferred to 3MM Whatman paper, dried and autoradiographed. DNA bending by GcvA6xhis was calculated as described by Kim et al. (1989)
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Ni+-NTA agarose affinity matrix (Qiagen) was dispensed into 30 µl aliquots for each co-ppt and washed twice with 400 µl loading buffer C to equilibrate, each time briefly centrifuging and decanting the supernatant with a Pasteur pipette. GcvA6xhis (bait) was diluted to 50 µg ml-1 in 200 µl loading buffer C, added to 30 µl affinity matrix and incubated with gentle mixing at 20 °C for 15 min. The affinity matrix was collected by centrifugation as above and the supernatant saved as sample 1. The Ni+-NTA agarose was washed twice with 200 µl loading buffer C to remove unbound protein. Following each of the washes the Ni+-NTA agarose was centrifuged, and the supernatants decanted and saved as samples 2 and 3. GcvR (target) was diluted to 50 µg ml-1 in 200 µl loading buffer C and added to the Ni+-NTA agaroseGcvA6xhis complex and incubation continued at 20 °C for 15 min. The affinity matrix was collected by centrifugation and the supernatant saved as sample 4. The matrix was then washed twice with 200 µl loading buffer C to remove unbound target protein. Following each of the washes the Ni+-NTA agarose was centrifuged, the supernatants decanted and saved as samples 5 and 6. The Ni+-NTA agaroseprotein complex was washed twice with 200 µl wash buffer B to remove target protein non-specifically bound to the matrix. Each wash was incubated for 5 min at 20 °C with gentle mixing, the mixtures centrifuged, and the supernatants collected as samples 7 and 8. The Ni+-NTA agarose-protein complex was then washed twice with 200 µl elution buffer. Each wash was incubated for 5 min at 20 °C with gentle mixing, the Ni+-NTA agarose was collected by centrifugation, and the supernatants collected and combined as sample 9. Four volumes of acetone were added to each sample, the proteins precipitated on ice for 30 min, and pelleted by centrifugation at 14000 r.p.m. [16000 g in a refrigerated (4 °C) Eppendorf microfuge]. The supernatants were decanted and the proteins dried under vacuum. The protein samples were resuspended in SDS loading buffer, heated at 95 °C for 5 min and loaded onto a 10% polyacrylamide/SDS gel.
Equilibrium dialysis.
Equilibrium dialysis was conducted using a micro-chamber equilibrium dialysis apparatus with pairs of 100 µl chambers separated by a 1200014000 molecular mass cut off Spectrapor dialysis membrane (Spectrum Medical Industries). Each protein tested was diluted to 25 µM in dialysis buffer (20 mM HEPES/NaOH pH 7·4, 150 mM NaCl, 0·1 mM EDTA, 10% glycerol) and placed in chambers (inside) opposite chambers containing [14C]glycine (16500 c.p.m.) in dialysis buffer (outside). Following overnight mixing of the dialysis cells at 20 °C, 50 µl samples from each chamber on opposite sides of the membrane were collected. The amount of [14C]glycine in each of the samples was determined by counting the samples in 10 ml Budget-Solve scintillation cocktail (Research Products International). The amount of [14C]glycine bound was determined by subtracting the counts outside from the counts inside each pair of chambers (Miller, 1992
).
Random mutagenesis.
Mutagenesis of gcvR was performed using a PCR mutagenesis protocol (Zhou et al., 1991 ). PCR products were generated using primers complementary to the DNA template outside of the gcvR gene and overlapping an EcoRI restriction site at the 5' end and a HindIII site at the 3' end. The PCR products were digested with EcoRI/HindIII, cloned into plasmid pBR322 and used to transform the gcvA mutant lysogen GS998
gcvT::lacZ carrying the gcvAR197G allele on the single-copy plasmid pGS311. The gcvAR197G allele results in constitutive expression of the gcv operon (Jourdan & Stauffer, 1998
). Transformants were selected on LB agar containing AP+TC to maintain both plasmids. Transformants were spotted onto GM agar supplemented with inosine, AP, TC, X-gal and TPEG. TPEG is a competitive inhibitor of ß-galactosidase and increases the stringency of the selection. When WT gcvA is carried on the single-copy plasmid and WT gcvR is carried on the multi-copy plasmid, colonies are white due to low levels of expression of the gcvT::lacZ fusion. Alternatively, when the gcvAR197G allele was expressed from the single-copy plasmid along with the WT gcvR allele on the multi-copy plasmid, colonies were blue due to high levels of expression of the gcvT::lacZ fusion. We assumed that mutations in gcvR that restore repression in the presence of the gcvAR197G allele would be less blue. Thus, light blue colonies, presumably with increased repression of gcvT::lacZ, were saved for further analysis.
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RESULTS |
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Initially we tested whether GcvA binding alone induces a bend in DNA. DNA fragments carrying GcvA binding sites 3+2+1 were cloned into the pBend2 vector (see Methods), and five different restriction enzymes were used to generate DNA fragments of the same length, but with the GcvA binding sites at different positions relative to the centre of the fragments (Fig. 2). Gel electrophoresis of the unbound fragments showed no significant intrinsic DNA bending (Fig. 3
). The addition of GcvA resulted in three shifted bands. Although we did not perform DNase I footprinting on the shifted complexes, previous studies showed that the three GcvA binding sites have affinities for GcvA in the order 2 3 1 (Wilson et al., 1995
). The different positions of the GcvA binding sites within the equal length fragments resulted in different relative mobilities of each of the three GcvA/DNA complexes (Fig. 3
). The apparent bend angle was calculated for the gcv control region using the formula µM/µE=cos
/2, where µM is the mobility of the DNA with the protein bound near the centre of the fragment and µE is the mobility for the DNA fragment with the binding site located near the end of the DNA (Kim et al., 1989
). When GcvA was bound to site 2 (band A in Fig. 3
), a bend angle of about 30° was calculated, when bound to sites 2+3 (band B in Fig. 3
) a bend angle of about 100° was calculated, and when bound to all three sites (band C in Fig. 3
), a bend angle of about 12° was calculated. One explanation for the loss of bending when all three sites are occupied is that a GcvA-mediated loop forms, the mobility being relatively independent of the position of the binding sites within the loop (Fig. 4
). Although other explanations are possible, we favour this interpretation based on results discussed below. When the GMS assay was performed on a DNA fragment carrying only GcvA site 1, a bend angle of about 75° was calculated (not shown). If phased with the bend induced at sites 3+2, a looped structure is possible. We also used a DNA fragment carrying the three GcvA binding sites but with a 4 bp change in site 1 that reduced the affinity of GcvA for site 1 about twofold and that resulted in a loss of GcvA-mediated repression (Wilson et al., 1995
; Wonderling et al., 2000
). The ability of GcvA to bind sites 2+3 and bend the DNA were unchanged (Fig. 5a
). However, when GcvA was bound to all three sites a bend angle of about 80° was calculated. Thus, although the bp changes only resulted in a small decrease in the ability of GcvA to bind to site 1, they significantly altered GcvA-induced bending of the DNA. We also inserted 1·5 and 2·5 turns of DNA between site 1 and sites 2+3. For both the +15 bp insert (not shown) and the +25 bp insert (Fig. 5b
), GcvA binding to sites 2+3 and its ability to bend DNA were essentially unchanged. However, when bound to all three sites, a bend angle of about 25° was calculated. If the 75° bend induced by GcvA bound to site 1 is phased opposite to the 100° bend induced by GcvA bound to sites 3+2, the data would be consistent with the bending pattern observed. The results are summarized in Fig. 4
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Does glycine directly affect GcvA-mediated expression of gcv?
Since glycine does not significantly affect GcvAs ability to bind or bend DNA, we wanted to determine how glycine is involved in the activation mechanism. It was reported previously that mutations in gcvR result in constitutive expression of the gcv operon, but a reduced growth rate (Ghrist & Stauffer, 1995 ). It is possible that the reduced growth rate is a result of glycine limitation due to its rapid catabolism by the GCV enzyme system. If true, this would suggest that activation of the gcv operon could occur in the absence of glycine and would be consistent with the GMS assay results. Therefore, we tested whether the reduced growth rate in the gcvR mutant strain GS1053 was due to altered glycine levels in the cell. The generation times of the WT strain GS162 grown in GM medium vs GM+glycine at 30 °C are 85 and 84 min, respectively, and for the gcvR strain GS1053 are 130 and 84 min, respectively. Thus, the gcvR mutation results in glycine auxotrophy, but constitutive expression of gcv. Growth of the gcvR strain in GM+inosine also reduced the generation time to 84 min. Since a glycine molecule is incorporated into the purine ring structure, the reduced generation time is possibly due to a sparing effect of glycine by purine supplementation.
We also constructed a glyA gcvR double mutant strain GS1089. This strain requires an exogenous source of glycine for growth due to the glyA mutation (Pizer, 1965 ). We used the Gly-Ser-Phe tripeptide to limit glycine (glycine concentration 50 µg ml-1). The generation time of the WT strain in GM+tripeptide at 30 °C was 82 min and the double mutant was more than 174 min. The addition of glycine to the medium reduced the generation time of the mutant to that of the WT, suggesting that the double mutant is starved for glycine. In the WT lysogen, gcvT::lacZ expression was threefold lower in the presence of the tripeptide compared to glycine, suggesting that the concentration of glycine as tripeptide is limiting for activation (Table 2
). In the glyA gcvR mutant lysogen, gcvT::lacZ expression was constitutive, although the cells were starved for glycine. These results and the results of the GMS assays suggest that glycine probably does not interact directly with GcvA to activate the gcv operon.
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DISCUSSION |
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Since glycine did not alter GcvAs ability to bind or bend gcv DNA, and since GcvR is required for GcvA to mediate negative regulation of the gcv operon, as well as for GcvA to show a positive response to glycine (Ghrist & Stauffer, 1995 ), it seemed possible that glycine might interact with GcvR rather than GcvA. We tested this possibility by equilibrium dialysis. The results showed that glycine binds GcvR rather than GcvA (Fig. 6
), suggesting that the co-activator glycine functions by a different mechanism for gcv control compared to the co-activators for other LTTR-regulated operons. Results from in vivo studies using a LexA two-hybrid system suggested that a direct interaction occurs between GcvA and GcvR (Ghrist et al., 2001
). The in vitro results from this study showed that when GcvA6xhis was bound to Ni+-NTA agarose affinity matrix, followed by addition of WT GcvR, a significant amount of GcvR co-eluted with GcvA during a high imidazole wash (Fig. 7a
). Furthermore, the addition of glycine (1 mM) to the loading and wash buffers inhibited this interaction (Fig. 7b
). These results are consistent with a direct GcvA/GcvR interaction and suggest that the role of the co-inducer glycine is either to block this association or to cause a dissociation of the complex once formed. The isolation of gcvR mutants that encode proteins with reduced abilities to bind glycine and that result in a reduced ability of glycine to activate gcvT::lacZ expression is consistent with this model. We used purified GcvR in a GMS assay with gcv DNA and in both the absence and presence of glycine. However, we could not show any binding of GcvR to gcv DNA (data not shown). These results suggests that GcvR interacts directly with GcvA to form a repression complex: a somewhat unconventional mechanism for prokaryotes (Fig. 8A
).
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It is worth noting that full repression of the gcv operon requires the addition of purines to GM medium (Wilson et al., 1993a ; Ghrist & Stauffer, 1995
). Although the mechanism of purine involvement is unknown, purines could either enhance the interaction between GcvA and GcvR, forming a more stable repressor complex, or change the conformation of the repressor complex at the promoter to prevent transcriptional initiation by RNAP. Furthermore, the addition of both glycine and purines to the growth medium results in derepression of the gcv operon (Stauffer & Stauffer, 1994
; Wilson et al., 1993a
). The dominance of glycine to purines in the regulatory mechanism, and the ability of glycine to disrupt the GcvA/GcvR interaction are consistent with a GcvA/GcvR complex being necessary for purine-mediated repression. Since all three GcvA binding sites are required for normal repression of the gcv operon (Wilson et al., 1995
; Wonderling et al., 2000
), it is likely that GcvA and GcvR form a repression loop (Fig. 8A
). DNA looping normally occurs between identical proteins (Gralla & Collado-Vides, 1996
). Verification of this as a heterologous protein system for DNA looping will provide a novel perspective into the basic mechanism by which protein/protein interactions control gene expression.
Previously it was shown that specific amino acid residues in the -subunit of RNAP (
-carboxy-terminal domain), when mutated, disrupt GcvA-mediated activation of the gcv operon, but not GcvA-mediated repression (Jourdan & Stauffer, 1999
). In addition, positive control mutants were isolated in gcvA that decreased GcvAs ability to activate gcv transcription, but that did not affect DNA binding or GcvA-mediated repression (Jourdan & Stauffer, 1998
). Since GcvA-mediated activation requires GcvA bound to sites 2 and 3, located 214271 bp upstream of the transcription start site, it is reasonable to assume that a looped nucleoprotein structure is required for an appropriate GcvA/RNAP contact for activation (Fig. 8B
). Although the results of this study show that GcvA also induces a bend in the DNA, it is not known whether the GcvA-induced DNA bending plays a direct role in GcvA-mediated regulation of the gcv operon.
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ACKNOWLEDGEMENTS |
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Received 9 January 2002;
revised 19 March 2002;
accepted 2 April 2002.
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