s2fieldsofstudy: Biology Chemistry

text:
The Significance of EXDD and RXKD Motif Conservation in Rel Proteins*

Monofunctional and bifunctional classes of Rel proteins catalyze pyrophosphoryl transfer from ATP to 3′-OH of GTP/GDP to synthesize (p)ppGpp, which is essential for normal microbial physiology and survival. Bifunctional proteins additionally catalyze the hydrolysis of (p)ppGpp. We have earlier demonstrated that although both catalyze identical the (p)ppGpp synthesis reaction, they exhibit a differential response to Mg2+ due to a unique charge reversal in the synthesis domain; an RXKD motif in the synthesis domain of bifunctional protein is substituted by an EXDD motif in that of the monofunctional proteins. Here, we show that these motifs also determine substrate specificities (GTP/GDP), cooperativity, and regulation of catalytic activities at the N-terminal region through the C-terminal region. Most importantly, a mutant bifunctional Rel carrying an EXDD instigates a novel catalytic reaction, resulting in the synthesis of pGpp by an independent hydrolysis of the 5′Pα-O-Pβ bond of GTP/GDP or (p)ppGpp. Further experiments with RelA from Escherichia coli wherein EXDD is naturally present also revealed the presence of pGpp, albeit at low levels. This work brings out the biological significance of RXKD/EXDD motif conservation in Rel proteins and reveals an additional catalytic activity for the monofunctional proteins, prompting an extensive investigation for the possible existence and role of pGpp in the biological system.

Monofunctional and bifunctional classes of Rel proteins catalyze pyrophosphoryl transfer from ATP to 3-OH of GTP/GDP to synthesize (p)ppGpp, which is essential for normal microbial physiology and survival. Bifunctional proteins additionally catalyze the hydrolysis of (p)ppGpp. We have earlier demonstrated that although both catalyze identical the (p)ppGpp synthesis reaction, they exhibit a differential response to Mg 2؉ due to a unique charge reversal in the synthesis domain; an RXKD motif in the synthesis domain of bifunctional protein is substituted by an EXDD motif in that of the monofunctional proteins. Here, we show that these motifs also determine substrate specificities (GTP/GDP), cooperativity, and regulation of catalytic activities at the N-terminal region through the C-terminal region. Most importantly, a mutant bifunctional Rel carrying an EXDD instigates a novel catalytic reaction, resulting in the synthesis of pGpp by an independent hydrolysis of the 5P ␣ -O-P ␤ bond of GTP/GDP or (p)ppGpp. Further experiments with RelA from Escherichia coli wherein EXDD is naturally present also revealed the presence of pGpp, albeit at low levels. This work brings out the biological significance of RXKD/EXDD motif conservation in Rel proteins and reveals an additional catalytic activity for the monofunctional proteins, prompting an extensive investigation for the possible existence and role of pGpp in the biological system.
The adaptability to changing environments determines the survival of an organism. Microorganisms utilize a hyperphosphorylated guanine nucleotide guanosine 5Ј-(tri-or di)phosphate,3Ј-diphosphate ((p)ppGpp), 6 also known as the "alarmone" to cope up with unfavorable environmental conditions. (p)ppGpp, through a phenomenon termed the "stringent response," accomplishes this by a rapid shutdown of active transcription, translation, and up-regulation of protein degradation and amino acid synthesis (1). The major effect of stringent response is so far attributed to the interactions of ppGpp to RNA polymerase, factors, and DksA (2,3). Apart from its effect on transcription and translation, (p)ppGpp is implicated in the regulation of a wide variety of physiological processes including sporulation, antibiotic production, nucleotide and fatty acid metabolism, surface organelle production, and more importantly, in the virulence of pathogenic organisms (4).
(p)ppGpp is synthesized and hydrolyzed by two distinct domains in the N-terminal region of the Rel family of proteins (5)(6)(7). They are further grouped into a Rel/SpoT bifunctional class (that synthesize and hydrolyze) and a RelA monofunctional class (that can only synthesize) (8). Although both domains are present in all Rel proteins, monofunctional proteins lack hydrolysis activity due to the absence of a conserved HDXXED motif in the hydrolysis domain (9). Although the synthesis and hydrolysis activities are confined to the N-terminal ϳ385 amino acids, the C-terminal region (ϳ385-750) ensures their regulation (10,11). In Streptococcus equisimilis (Rel S. eq ), a bifunctional protein, the C-terminal region renders a negative regulatory effect on the synthesis activity and facilitates hydrolysis; deletion of the C-terminal region enhances synthesis activity by ϳ12-fold while significantly reducing the hydrolysis activity (ϳ150-fold) (11). A direct interaction of C-terminal region with the synthesis domain has also been reported (10); however, the key players remain elusive.
During stress, Rel proteins catalyze the transfer of a pyrophosphate from ATP to the 3Ј-OH of GTP or GDP to synthesize pppGpp or ppGpp, respectively, collectively termed as (p)ppGpp (7,12,13). As favorable conditions are restored, the stringent response can be reversed by the hydrolysis of (p)ppGpp to GTP/GDP and pyrophosphate (PP i ) by the bifunctional Rel proteins. Although both GTP and GDP can act as pyrophosphate acceptors for (p)ppGpp synthesis, the Rel proteins seem to display differential preference for either GDP or GTP; Rel from Rel S. eq prefers GTP over GDP (11), and Rel from Mycobacterium tuberculosis (Rel M. tb ) (14) and RelA from Escherichia coli (RelA E. coli ) (15) are reported to utilize both GTP and GDP with equal efficiency. However, as the intracellular concentration of GDP is very low, it is believed that GTP would be the principal pyrophosphate acceptor (1), and this notion is substantiated by the presence of phosphatases like GppA that hydrolyze pppGpp to ppGpp (16,17). On the other hand, in E. coli ppGpp has been shown to be the most potent molecule to elicit stringent response (1, 4), which would imply a preference for GDP. In line with this, a closer analysis revealed that differences in (p)ppGpp synthesis by RelA E. coli and Rel M. tb , reported in our earlier work, are not in consensus with the aforesaid equal preference for GTP and GDP.
Earlier, using the N-terminal regions of Rel proteins, we had shown that they differ in utilizing Mg 2ϩ for (p)ppGpp synthesis and had attributed this difference to a charge reversal in the synthesis domain where an RXKD in the bifunctional protein is substituted to an EXDD in monofunctional proteins (8). Here, using the full-length proteins, we further explore the significance of the distinct conservation of these motifs in Rel proteins. In contrast to the earlier reports, we found that monofunctional RelA E. coli utilizes GDP and bifunctional Rel M. tb utilizes GTP as the principal pyrophosphate acceptor, and this specificity is determined by the EXDD and RXKD motifs, respectively. We further find that the presence of an RXKD motif also leads to cooperative nucleotide binding, whereas EXDD does not. Interestingly, in bifunctional protein the substitution RXKD 3 EXDD (in Rel M. tb ) led to a drastic reduction in (p)ppGpp synthesis (where 3 indicates the interchange of the motifs). In contrast, a similar reversal in monofunctional proteins (i.e. EXDD 3 RXKD in RelA E. coli ) resulted in enhanced synthesis. Analogous effects were not found when the N-terminal regions of the proteins were employed (8), implying a critical role for these motifs in determining the regulation of catalytic activities through their interaction with the C-terminal region. The most important finding, however, is that RXKD 3 EXDD substitution in the bifunctional Rel M. tb resulted in the synthesis of a novel molecule that we identify as pGpp. Inspired by this observation, we probed the ability of RelA E. coli , which naturally carries an EXDD motif, to synthesize pGpp. The presence of pGpp in this reaction, albeit at low levels, opens the avenue to explore the significance of pGpp versus (p)ppGpp in microbial physiology.
Protein expression was carried out in E. coli. BL21 pLys cells from 2 liters of growth media were resuspended in 50 mM Tris-HCl (pH 8), 300 mM NaCl, 1 mM protease inhibitor mixture (Sigma), 1 mM dithiothreitol, 1% Triton X-100 and 10% glycerol. Cells were lysed by sonication. The supernatant was collected by centrifugation at 35,000 relative centrifugal force for 30 min followed by loading onto a nickel-nitrilotriacetic acid column (Amersham Biosciences). The column was washed with 10 column volumes of wash buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM dithiothreitol, and 50 mM imidazole. The protein was eluted using a gradient of 20 col-umn volumes (0 -0.5 M) of imidazole, and the protein eluted at 40% of the gradient. The purified protein was then subjected to gel-filtration chromatography (Superdex 200, Amersham Biosciences). The buffer was exchanged with 50 mM HEPES (pH 8), 150 mM NaCl, and 1 mM dithiothreitol. The monomer fraction of the protein was collected, which had a final concentration of 0.3-0.5 mg/ml and was stored at Ϫ80°C after snap-freezing in liquid N 2 . The total yield was 0.5 mg.
(p)ppGpp Synthesis Assay-pppGpp/ppGpp synthesis assays were carried out as described earlier (8). A 5-l reaction volume contained 50 mM HEPES (pH 8.0), 100 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl 2 for RXKD-containing proteins, 50 mM for EXDD-containing proteins (8), 5 mM GTP/GDP, 5 mM ATP, 1 Ci of [␥-32 P]ATP, and 5 M WT or MT-Rel proteins at 37°C for 10 -30 min. ATP and GTP/GDP were varied from 0 to 20 mM (with corresponding variations in Mg 2ϩ concentrations) for cooperative ATP and GTP/GDP binding studies. The reactions were stopped by adding 1 l of 6 M formic acid. The mixture was then centrifuged at 13,000 rpm for 10 min. 5 l of the sample was spotted on the polyethyleneimine-coated TLC (Merck), resolved in 1.5 M KH 2 PO 4 (pH 3.4) buffer, and subjected to autoradiography to detect the formation of (p)ppGpp. For quantitation, the spots corresponding to (p)ppGpp were cored out from polyethyleneimine-coated TLC, and the counts were determined.
To determine the kinetic constants for GTP/GDP, reactions were carried out as described above with varying concentrations of GTP/GDP (0 -15 mM), 15 mM ATP for 10 min at 37°C. For the RXKD-containing proteins, Mg 2ϩ concentration was equal to that of the total nucleotides and for EXDD-containing proteins, 5-fold higher concentrations were used. 1 Ci of [␥-32 P]ATP/reaction was used as tracer for determining concentrations of the product. K m and V max values were determined by nonlinear regression analysis carried out with Graph-Pad prism software.
pGpp Synthesis Assay-The efficiency of MT-Rel M. tb to synthesize pGpp when GTP and GMP are provided as substrates was compared in Fig. 4C. For these, synthesis reactions were carried out as above using 5 mM GTP/GMP as the substrate along with 5 mM ATP and 1 Ci of [␥-32 P]ATP and spotted on a polyethyleneimine-TLC. For quantitation, the spots corresponding to pGpp were cored out from polyethyleneimine-TLC, and the counts were determined.
pppGpp Hydrolysis Assay-pppGpp hydrolysis assays were carried out in 5-l reaction volumes containing 50 mM HEPES (pH 8.0), 100 mM NaCl, 1 mM dithiothreitol, 10 mM MnCl 2 , 5 mM pppGpp 32 , and 5 M WT or MT-Rel M. tb proteins at 37°C for 30 min. The reaction was stopped by adding 1 l of 6 M formic acid. The mixture was then centrifuged at 13,000 rpm for 10 min. 5 l of the sample was spotted on the polyethyleneimine-coated TLC (Merck), resolved in 1.5 M KH 2 PO 4 (pH 3.4) buffer, and subjected to autoradiography to detect the release of PP i . For quantitation, the spots corresponding to PP i were cored out from polyethyleneimine-coated TLC, and the counts were determined.
Intrinsic Chemical Stability of the New Product-MT-Rel M. tb was employed in a (p)ppGpp synthesis assay, and the stability of the new product (see "Results") formed, pGpp, was tested for its identity from its isoform, ppGp. After the synthesis reaction for 30 min, the enzyme was heat-inactivated, and 1 l of 2 N NaOH was added to 5 l of the reaction. It was spotted on a polyethyleneimine-coated TLC (as above) together with a negative control devoid of NaOH.
Site-directed Mutagenesis-Site-directed mutagenesis was carried out by overlapping PCR method as described earlier (8).
The following forward and reverse primers were used, with desired change in the codon sequence. In Rel M. tb Arg-348 and Lys-350 were mutated to Glu and Asp, respectively (Rel M. tb R348E/K350D forward primer, 5Ј-ATGGCGGGTGAGT-TCGACGACTACATCGC-3Ј; Rel M. tb R348E/K350D reverse primer 5Ј-GCGATGTAGTCGTCGAACTCACCCGCCATC-3Ј). In RelA E. coli Glu-306 and Asp-308 were mutated to Arg and Lys, respectively (RelA E. coli E306R/D308K forward primer, 5Ј-CGCCACCTGCCCGGGCGGTTTAAGGATT-ACGTCGC-3Ј; RelA E. coli E306R/D308K, reverse primer, 5Ј-GCGACGTAATCCTTAAACCGCCCGGGCAGGTG-GCG-3Ј). In each case, the fragments were amplified (Pfu DNA polymerase) using the forward primer of the gene and the reverse primer containing the mutation and the reverse primer of the gene and the forward primer with the mutation. Amplified fragments were gel-purified. Equal quantities of both fragments were used as template for the full-length amplification of the gene with mutation using the initial primers. The PCR-amplified mutant gene was digested with NdeI and Hin-dIII and cloned into corresponding sites in pET-28 vector. The mutations were confirmed by sequencing.
Intrinsic Tryptophan Fluorescence-Tryptophan fluorescence studies were carried out using LS 55 Fluorescence Spectrometer (PerkinElmer Life Sciences) at room temperature. The protein was monitored with an excitation wavelength of 280 nm (slit width of 2.5 nm) and emission wavelength of 300 -500 nm (slit width of 5 nm).

RelA E. coli Utilizes GDP as the Primary Pyrophosphate
Acceptor-In our earlier work, we reported a differential effect of Mg 2ϩ on (p)ppGpp synthesis by the N-terminal fragments of monofunctional and bifunctional Rel proteins (8). When synthesis reactions were carried out using the N-terminal regions of RelA E. coli and mutant Rel M. tb (RXKD 3 EXDD) with ATP and GTP as substrates, we were surprised to observe an additional spot in the autoradiograms apart from pppGpp (8). Here, the same assay was performed using full-length RelA E. coli (WT-RelA E. coli ). Like the N-terminal fragments, it also showed an additional spot in the autoradiograms (Fig. 1, dotted circle in lane 1) apart from the anticipated pppGpp (pentaphosphate) (lane 1, Fig. 1). Based on its position in polyethyleneiminecoated TLC, the additional spot appeared to be the tetraphosphate, ppGpp. To confirm this GTP was replaced by GDP, which resulted in the formation of ppGpp (tetraphosphate) (lane 2, Fig. 1). The position of the additional spot indeed corresponds to the ppGpp produced in lane 2. However, ppGpp synthesis in lane 1 was intriguing, as no GDP was provided in the reaction except a possible contaminant in the GTP pool that might arise from the intrinsic hydrolysis of GTP. The formation of ppGpp utilizing even the trace amount of contaminant GDP would indicate a very high affinity of RelA E. coli for GDP. This rationale would be contrary to the earlier reports that suggested equal affinity of WT-RelA E. coli for GTP and GDP (15). Interestingly, the additional spot could also be detected with the N-terminal fragment of mutant Rel M. tb that had an EXDD motif (8) like WT-RelA E. coli . Taken together, these results indicated EXDD-containing proteins to have a higher affinity for GDP than GTP. On the other hand, Rel from S. equisimilis (Rel S. eq ) was reported to have a higher affinity for GTP over GDP (11), and Rel M. tb was reported to utilize GTP and GDP with equal efficiency (14). Interestingly, both Rel M. tb (14) and Rel S. eq (11) are bifunctional proteins with an RXKD motif. To clarify the role of these motifs, if any, in dictating the specificities for GTP and GDP, the following experiments were conducted.
EXDD and RXKD Motifs Determine Substrate Specificity and Nucleotide Binding-To examine the specificities for GTP/ GDP and the role of EXDD/RXKD motifs in governing this specificity, we used four Rel proteins; two wild types (WT-RelA E. coli with an EXDD and WT-Rel M. tb with an RXKD) and their mutants where the motifs were interchanged (MT-RelA E. coli with EXDD 3 RXKD and MT-Rel M. tb with RXKD 3 EXDD). Each protein was subjected to an independent synthesis reaction using ATP and either GTP or GDP as substrates. The highest amount of product synthesized (pppGpp when GTP is the substrate and ppGpp when GDP is the substrate) by each protein was taken as 100% as shown in Fig. 2A. Here, RXKD-containing proteins (WT-Rel M. tb and MT-RelA E. coli ) show a distinct preference for GTP over GDP, whereas the EXDD containing proteins (WT-RelA E. coli and MT-Rel M. tb ) conversely prefer GDP over GTP. The preference for GTP/ GDP by these motifs is further substantiated by the kinetic constants, as shown in Table 1. GDP is preferred over GTP by WT-RelA E. coli (EXDD) with a lower K m and a higher V max , reflected in ϳ15-fold higher catalytic efficiency (V max /K m ) for GDP over GTP. On the other hand, RXKD-containing WT-Rel M. tb and MT-RelA E. coli show a preference for GTP over GDP, shown by their lower K m and higher V max for GTP. These values are not in agreement with the kinetic constants reported earlier (5,15) for the wild type proteins RelA E. coli and Rel M. tb , as they were determined in the presence of cofactors (mRNA, tRNA, and/or ribosome) or denaturants (methanol or detergents) that were absent in our study. Nevertheless, these results demonstrate the importance of the motifs RXKD/EXDD in governing substrate specificities in Rel proteins.
It appears that the monofunctional RelA E. coli prefers GDP over GTP, and the bifunctional Rel M. tb prefers GTP over GDP, like Rel S. eq . As co-operative ATP binding was reported for Rel S. eq (11), we examined the same for Rel M. tb and RelA E. coli to investigate the effect of the motifs RXKD/EXDD, if any. The aforesaid (p)ppGpp synthesis assays were carried out to examine cooperativity in nucleotide binding. Percent (p)ppGpp synthesized (% activity) was plotted against increasing ATP (Fig.  2B) and GTP (Fig. 2C) concentrations for WT-RelA E. coli , MT-RelA E. coli , and WT-Rel M. tb . WT-Rel M. tb , like Rel S. eq (11), having an RXKD motif, also exhibited a cooperative ATP binding as suggested by the sigmoidal nature of the curve (Fig. 2B). Similarly, cooperative GTP binding also was observed for WT-Rel M. tb (Fig. 2C). WT-RelA E. coli , with an EXDD motif, on the other hand, displayed a hyperbolic behavior indicating the absence of cooperativity for both ATP and GTP binding. However, the hyperbolic behavior was reversed to sigmoidal behavior when MT-RelA E. coli , having an RXKD motif, was employed (Fig. 2, B and  C). A reversal to hyperbolic nature in MT-Rel M. tb , with an EXDD motif, could not be shown, as the activity of the protein was compromised significantly (see below). These experiments indicate that RXKD motif, but not EXDD, renders cooperative nucleotide binding.

Effect of Interchanging EXDD and RXKD on (p)ppGpp Synthesis-To
understand the effect of interchanging the motifs, the four proteins mentioned above were used to analyze their catalytic activities. (p)ppGpp synthesis and hydrolysis reactions were carried out as described under "Experimental Procedures." Percent (p)ppGpp synthesized by these proteins utilizing GDP (for EXDD proteins) and GTP (for RXKD proteins) is shown in Fig. 3A. Intriguingly, MT-RelA E. coli (EXDD 3 RXKD) showed an enhanced synthesis (ϳ2.5-fold) compared with WT-RelA E. coli , whereas MT-Rel M. tb (RXKD 3 EXDD) displayed a drastic reduction (ϳ90% of WT-Rel M. tb ) (Fig. 3A) without significantly affecting (p)ppGpp hydrolysis (data not shown). The contrasting behavior of these mutants led us to FIGURE 2. A, effect of RXKD and EXDD motifs in determining specificities for GTP and GDP. Independent (p)ppGpp synthesis assays were carried out as in Fig. 1 using the proteins and either GTP or GDP along with [␥-32 P]ATP, as indicated, and % activity is shown. The highest amount of product synthesized (pppGpp when GTP as substrate and ppGpp when GDP as the substrate) by each protein was taken as 100%. B and C, RXKD motif renders cooperative ATP (B) and GTP (C) binding. % (p)ppGpp synthesized by different Rel proteins using GDP for EXDD-and GTP for RXKD-containing proteins is plotted against varying ATP (B). Similarly, in C, GTP or GDP was varied for RXKD-and EXDD-containing proteins, respectively.

TABLE 1 The kinetic constants for GTP and GDP in RXKD and EXDD containing Rel proteins
All the values were determined in the absence of cofactors such as mRNA, tRNA, and/or ribosomes or denaturants such as methanol and detergents, unlike the earlier studies (5,15). Values obtained from three independent experiments were used for calculating the S.D. MT-Rel M. tb (EXDD) was not used here, as (p)ppGpp synthesis was reduced by 90% (see Fig. 3A). probe if any conformational change in the protein affected the synthesis activity. This was examined utilizing the intrinsic tryptophan fluorescence. A major difference in the fluorescence spectra was seen for WT-RelA E. coli versus MT-RelA E. coli (Fig. 3B). But on the contrary, Rel M. tb proteins (WT-Rel M. tb and MT-Rel M. tb ) did not exhibit any difference (Fig. 3C). As EXDD 3 RXKD in MT-RelA E. coli led to an enhanced (ϳ2.5-fold) synthesis and reduced fluorescence, we wanted to discern the candidate tryptophan that contributed significantly toward the reduced fluorescence. A sequence-based structural comparison with Rel S. eq identified Trp-260 (see the inset, Fig. 3, B and C) as a potential residue based on its position in the catalytic loop. This loop is believed to play an important role in regulating the synthesis activity (8,18). W260A mutant was, thus, prepared to examine its role, and as expected, the mutation resulted in a drastic reduction in intrinsic fluorescence (Fig. 3B), suggesting Trp-260 as the major contributor to the intrinsic fluorescence of WT-RelA E. coli . The implications of these observations are discussed under "Discussion."

Protein (motif)
The Presence of EXDD Generates a New Catalytic Site-A reduction in the synthesis activity by MT-Rel M. tb was unanticipated. However, we were surprised by the presence of a new spot (circled in lanes 4 and 5; Fig. 4A) in the autoradiograms of (p)ppGpp synthesis reactions catalyzed by MT-Rel M. tb . The new spot was not seen when WT-Rel M. tb (lanes 2 and 3) or the N-terminal domains (8) were used, and its migration was similar to that of [␥-32 P]GTP (lane 6, Fig. 4A). Mobilities similar to GTP indicated that the new product might contain identical number of phosphates on the guanine nucleoside, suggesting pGpp, ppGp, or pppG as the possible molecules. Because [␥-32 P]ATP was used in lanes 4 and 5, where the new molecule is formed, it must bear a radiolabeled pyrophosphate transferred from [␥-32 P]ATP to the 3Ј-OH of the guanine nucleotide. This eliminates ppGp and pppG as candidates, and hence, the product should be 5Ј-pGpp-3Ј. This was further examined by intrinsic (in)stability of the new product under alkaline conditions. This is because the presence of ␤-phosphates at the 3Ј-OH is known to be alkali-labile (19). In Fig. 4B, the new product (circled in lane 1) disappears when the reaction was subjected to alkaline conditions (lane 2), indicating that the new product bears a radiolabeled 3Ј-␤ phosphate. Hence the new product would be 5Ј-pGpp-3Ј and not its isoform, 5Ј-ppGp-3Ј, which is stable under alkaline conditions (19 -21).
Interestingly, the formation of pGpp requires GMP, which was not supplied. We hypothesized that the GMP part of pGpp would have arisen either as a contaminant in GTP/GDP pool or as a result of an ester hydrolysis between P ␣ and P ␤ of GTP/ GDP, perhaps due to the formation of a novel catalytic center in MT-Rel M. tb . However, Rel proteins were shown to utilize GMP very inefficiently (11,22), and because GTP/GDP, the efficient substrates, were provided in excess, the possibility of pGpp production due to contaminant GMP might be negated. This was further substantiated by comparing the efficiency with which MT-Rel M. tb utilizes GTP and GMP for pGpp synthesis, which is shown in lanes 2 and 3 of Fig. 4C, respectively. The amount of pGpp produced in these clearly depicts that GMP is a poor substrate for MT-Rel M. tb as compared with GTP, ruling out the possibility of utilizing contaminant GMP from the GTP/GDP pool to synthesize pGpp.
To verify the possibility that pGpp is formed due to a hydrolysis of the P ␣ -O-P ␤ bond of GTP/GDP, GMPCPP, a GTP analogue with a carbon in place of an oxygen between P ␣ and P ␤ was used to hinder the aforesaid ester hydrolysis (lane 7, Fig.  4A). Indeed, lane 7 shows the synthesis of pp(c)pGpp, but not pGpp, emphasizing the need for a hydrolysable oxygen between P ␣ and P ␤ of GTP/GDP to synthesize pGpp. Hence, pGpp was synthesized due to the aforesaid novel catalytic center, which was further substantiated by the release of a pyrophosphate from [␥-32 P]GTP (Fig. 5), as discussed below. It appears that introducing EXDD resulted in the formation of a new catalytic site to synthesize pGpp by either hydrolyzing GTP/GDP to GMP, which then accepts a pyrophosphate from ATP, or by hydrolyzing (p)ppGpp to pGpp. A critical role for the C-terminal region in catalyzing this reaction was brought out by the fact that only the full-length protein (carrying EXDD mutation) can synthesize pGpp. Therefore, the reaction catalyzed by fulllength WT-RelA E. coli , possessing an EXDD, was also examined for the presence of pGpp. The autoradiogram in Fig. 4D, showing a spot at an identical position (lane 2), confirmed that it also synthesized pGpp, although the amounts are insignificant compared with the amount of (p)ppGpp. As anticipated, pGpp synthesis was abolished by MT-RelA E. coli , with EXDD 3 RXKD substitution (lane 3, Fig. 4D), reiterating the need of EXDD for this activity. Further experiments were designed to understand this novel catalytic subsite.
The New Catalytic Subsite Functions Independent of the Pyrophosphoryl Transfer-Our results clearly demonstrate that EXDD motif along with the C-terminal region is capable of synthesizing pGpp, likely due to the hydrolysis of P ␣ -O-P ␤ bond of GTP/GDP. However, to determine the (in)dependence of this reaction on the pyrophosphate transfer from ATP, a nonhydrolysable ATP analogue, AMP-CPP, was used to inhibit the same. In reactions where [␥-32 P]GTP or [␥-32 P]ATP alone was used as substrates, no additional spots were detected (lanes 1  and 3, Fig. 5). Interestingly, complementing [␥-32 P]GTP with AMP-CPP led to the formation of labeled PP i (lane 2, Fig. 5), suggesting the hydrolysis of P ␣ -O-P ␤ bond of GTP even in the absence of a pyrophosphate transfer from ATP. This demonstrated that the new catalytic subsite in the full-length protein, created by EXDD, functioned independent of pyrophosphate transfer from ATP but required ATP binding at the active site.

DISCUSSION
The Rel family of proteins is essential for microbial survival under stress by virtue of its ability to metabolize (p)ppGpp, the mediator of stringent response (1,4,6,7,13). That monofunctional and bifunctional Rel proteins, due to a unique charge reversal in the synthesis domain, differ in Mg 2ϩ utilization to synthesize (p)ppGpp, was earlier reported by us (8). Driven by the observation that monofunctional proteins generate both pppGpp and ppGpp, although only GTP but not GDP was provided, together with ATP in the synthesis reaction, we set out to examine the effects rendered by the EXDD motif. Here, we uncover several other intriguing aspects of this seemingly simple charge reversal.
Here, for the first time we generalize substrate specificities for the Rel family of proteins based on the presence of a conserved motif in the nucleotide binding region of the synthesis domains. Proteins with an RXKD motif prefer GTP, and those with an EXDD prefer GDP as the principal pyrophosphate acceptor to synthesize (p)ppGpp. This preference is also indicated by the kinetic constants shown in Table 1. Although these values were determined in the absence of any cofactors or denaturants, unlike in the earlier reports (5, 15), we believe that RXKD and EXDD motifs continue to govern substrate specificity, although the absolute values of K m and V max would change in presence of the cofactors in vivo. So far, based on qualitative analysis, Rel M. tb was inferred to utilize GTP and GDP with equal efficiency (14), but a quantitative study on Rel S. eq showed a preference for GTP (11). Both being bifunctional RXKD-containing proteins, the motif-based generalization of specificity concurs with our finding that Rel M. tb also prefers GTP (Fig. 2). That RelA E. coli with an EXDD motif utilizes GDP (Fig. 2) as the principal pyrophosphate acceptor is also in concurrence. However, it is not in agreement with the equal GTP/GDP efficiencies reported by Cochran and Byrne (15). Perhaps this discrepancy arises due to the methods used to prepare RelA E. coli ; Cochran and Byrne (15) used the NH 4 Cl wash of 70 S ribosomes that also contained ribosome-associated GTPases (23), which would hydrolyze GTP used in the assays to GDP, leading to an apparent equal affinity. Also, the efficiency was calculated based on the total amount of both pppGpp and ppGpp synthesized. On the other hand, the procedures employed by us (see "Experimental Procedures") ensured high purity of the protein.
The implication of a high GDP affinity of RelA E. coli indicates its ability to synthesize ppGpp directly bypassing phosphatases GppA to hydrolyze pppGpp to ppGpp; ppGpp is known to be the major mediator of stringent response in E. coli (1,4). In addition, this high GDP affinity may also be utilized by RelA E. coli to ensure a rapid supply of ppGpp necessary to maintain a high fidelity of amino acid biosynthesis (24,25) and to elicit a prompt response to the dynamically changing environment. This is possible because active translation utilizes a large amount of GTP to ensure high fidelity (26,27) and that may perhaps result in a higher local GDP concentration in the microenvironment around the ribosomes which could be exploited by RelA E. coli .
Although significance to a high GDP affinity of RelA E. coli may be construed, the genesis of the differential GDP/GTP specificities of EXDD/RXKD motifs is intriguing. In the earlier work, based on the crystal structure of Rel S. eq (18) and Mg 2ϩindependent nucleotide binding in RXKD-containing proteins, we speculated that the phosphate groups of GTP could be coordinated by the positively charged Lys and Arg residues (8). Hence, the presence of ␥-phosphate may explain the observed preference of RXKD for GTP. In the case of EXDD enzymes, we reasoned that the interactions provided by Lys and Arg of RXKD are compensated by interactions from an additional Mg 2ϩ , coordinated by the carboxyl groups of Glu and Asp of EXDD (8). They may bind guanine nucleotides by replacing the Mg 2ϩ of GDP⅐Mg 2ϩ /GTP⅐Mg 2ϩ complexes with the EXDDcoordinated Mg 2ϩ . A possible reasoning for the preference of these motifs for GTP/GDP may stem from their different modes of nucleotide binding and also from the difference in coordination of Mg 2ϩ in GDP⅐Mg 2ϩ and GTP⅐Mg 2ϩ complexes. Mg 2ϩ of GDP⅐Mg 2ϩ coordinates P ␣ and P ␤ , and that in GTP⅐Mg 2ϩ would also involve P ␥ , leading to a difference in the positioning of Mg 2ϩ while binding to the protein. Such a difference in the position of Mg 2ϩ is observed in the structures of ppGpp bound to RNA polymerase (2,3) and GTP bound to Ras (28,29). This differential positioning of Mg 2ϩ may lead to differences in binding energy for motifs RXKD and EXDD to replace the GTP/GDP coordinated Mg 2ϩ such that it is optimal for EXDD to bind GDP⅐Mg 2ϩ and RXKD to bind GTP⅐Mg 2ϩ . Interestingly, K ϩ ion channels similarly exploit subtle differences in hydration energy to selectively transport K ϩ ions, but not Na ϩ , although the latter is smaller in size (30). Although this speculation may explain the differential specificities in Rel proteins, evidently rigorous crystal structure analysis in the presence of GDP and GTP for both EXDD and RXKD-containing proteins will be needed to comprehensively understand this intriguing attribute.
Apart from substrate specificity, different modes of regulation in bifunctional and monofunctional Rel proteins may be inferred based on the finding that RXKD imparts cooperative ATP and GTP binding but not EXDD (Fig. 2, B and C). Perhaps a better regulation is achieved by RXKD for bifunctional proteins as they need to regulate two activities (i.e. synthesis and hydrolysis) at distinct domains. Delving further, we found that the C-terminal region exerts a negative regulatory effect on (p)ppGpp synthesis by means of interactions mediated through these motifs at the N-terminal region. RXKD 3 EXDD reversal in bifunctional Rel M. tb led to a drastic reduction in (p)ppGpp synthesis, whereas EXDD 3 RXKD in RelA E. coli increased synthesis by 2.5-fold (Fig. 3A). However, when only the N-terminal regions of the same proteins were employed, a similar influence was not observed (8). Hence, the observed differences in synthesis are likely due to differences in interactions of the C-terminal region with the EXDD and RXKD motifs. An EXDD 3 RXKD interchange in MT-RelA E. coli apart from enhancing synthesis activity, exhibited a reduction in the intrinsic fluorescence (Fig. 3B). We attributed the reduced fluorescence in the full-length protein to a possible exposure of Trp-260 (in the catalytic loop) to an aqueous environment that was otherwise buried due to interactions by the C-terminal region. The presence of RXKD in the mutant would have altered the interactions between the synthesis domain and the C-terminal region and thereby enhanced synthesis. On the contrary, RXKD 3 EXDD in Rel M. tb that displayed a severe reduction in synthesis did not show a difference in fluorescence, although here too the catalytic loop contained a tryptophan (see the inset, Fig. 3C). Unaltered fluorescence negates a major conformational change, and the reduction in (p)ppGpp synthesis would perhaps have arisen due to a strengthened interaction of EXDD with the C-terminal region in the mutant. Such strengthening may not influence the environment of the tryptophan in the catalytic loop but would be necessary to occlude water to facilitate the formation of a new catalytic site. Together with the observed increase in synthesis for Rel S. eq (with RXKD) upon deleting the C-terminal region (11), we conclude that the C-terminal region regulates the activities of Rel proteins through interactions mediated by these motifs.
However, it would be interesting to compare the conformational changes seen in the full-length protein and the N-terminal half carrying only the catalytic domains (8). In the latter we had earlier proposed a "loop to helix" transition of the catalytic loop, present in the synthesis domain. Based on circular dichroism experiments, we had proposed that these structural changes occur in RXKD-containing Rel proteins (N-terminal part) with increasing Mg 2ϩ (8). Here, although the observed change in intrinsic fluorescence is due to a Trp residue present in the same catalytic loop, the conformational changes appear to arise due to an interaction between the C-terminal region in the full-length protein and the EXDD motif, as discussed above.
Thus far our work reveals several interesting features of Rel proteins, attributed by the motifs RXKD and EXDD. However, an important finding is the additional catalytic reaction by EXDD-containing proteins to synthesize pGpp (Fig. 4). This new catalytic activity, involving the hydrolysis of the 5Ј ␣-␤ ester bond of GTP/GDP/(p)ppGpp, occurs in same active site of the synthesis domain (Fig. 5). The key features of this additional reaction are as follows. 1) It requires the presence of an EXDD motif. 2) The reaction is independent of the pyrophosphate transfer reaction from ATP. 3) It requires both GTP/GDP and ATP bind to the pocket. 4) In addition, it also requires the C-terminal region, as the additional product, pGpp, was absent when the N-terminal regions alone were employed (8). The fact that pGpp synthesis was pronounced in RXKD 3 EXDD mutant of Rel M. tb than in RelA E. coli , where EXDD is present naturally, made it possible to identify the existence of pGpp. A possible reason for the different amounts of pGpp produced may be provided based on the strength of interactions between the C-terminal region and the synthesis domain as described above; the stronger the interaction, the lower the (p)ppGpp synthesized and higher the pGpp synthesized. These conjectures raise several possibilities and warrant the structure determination of full-length Rel proteins.
In summary, our findings open new avenues to further explore the function of Rel proteins. It may appear that pGpp is a byproduct of (p)ppGpp synthesis by RelA E. coli . However one cannot rule out that depending on the biological context, RelA E. coli may fine-tune interactions between synthesis domain and C-terminal region to promote pGpp synthesis. Thus, Gram-negative bacteria may have an evolutionary advantage of having a monofunctional RelA in addition to SpoT, a bifunctional enzyme, to adjust to the dynamic environment more rapidly than the Gram positives, which need to cope with a single copy of bifunctional Rel. As RXKD 3 EXDD in bifunctional proteins introduced a third activity, i.e. pGpp synthesis, efficient regulation of three independent catalytic activities in a single polypeptide chain may not be viable and, hence, the absence of an EXDD in bifunctional proteins. This would be in line with the drastic reduction in (p)ppGpp synthesis upon RXKD 3 EXDD substitution in bifunctional Rel M. tb . Similarly, monofunctional proteins would have overcome this constraint of regulating three activities by compromising the hydrolysis activity, ensured by the presence of SpoT in the organism and thereby facilitating the efficient acquisition of a new catalytic activity in the synthesis domain.


Transcription regulation of restriction-modification system Ecl18kI

Restriction-modification (R-M) system Ecl18kI is representative of R-M systems whose coordinated transcription is achieved through a separate DNA-binding domain of the methyltransferase. M.Ecl18kI recognizes an operator sequence located in the noncoding region that separates the divergently transcribed R and M genes. Here we show that, contrary to previous predictions, the two ecl18kI promoters are not divergent, but actually face one another. The binding of M.Ecl18kI to its operator prevents RNA polymerase (RNAP) binding to the M promoter by steric exclusion, but has no direct effect on RNAP interaction with the R promoter. The start point for R transcription is located outside of the intergenic region, opposite the initiation codon of the M gene. Regulated transcription of the potentially toxic ecl18kI R gene is accomplished (i) at the stage of promoter complex formation, through direct competition from complexes formed at the M promoter, and (ii) at the stage of promoter clearance, since R promoter-bound RNAP escapes the promoter more slowly than RNAP bound to the M promoter.

INTRODUCTION
Enterobacter cloacae type II restriction-modification (R-M) system Ecl18kI is naturally carried on plasmid pECL18 (1). Ecl18kI consists of two divergently transcribed genes, one coding for restriction endonuclease (R), and another coding for methyltransferase (M). The two genes are separated by an intergenic region of 109 bp. Ecl18kI is virtually identical to the better-studied SsoII system from Shigella sonnei: the two systems differ from each other in only 8 nt positions over their entire length; their intergenic regions are identical. In both systems, the intergenic region contains an inverted repeat AGGACAATTTGTCCT separated by a T at the center of symmetry. Coordinated expression of the ssoII genes is achieved through specific binding of the methyltransferase to this inverted repeat, called the operator (2). The operator is distinct from the SsoII recognition site 5 0 -CCNGG-3 0 and is recognized by a helix-turn-helix motif located at the N-terminus of the methyltransferase polypeptide (2). DNase I footprinting experiments demonstrate that M.SsoII protects a 48-52-bp region immediately upstream of its coding sequence (2). The region includes predicted consensus elements of the ssoII.M promoter, suggesting that the binding of M.SsoII autogeneously regulates its own synthesis by sterically excluding RNAP from the promoter.
While transcription regulation of SsoII was never explicitly studied, previous work assumed that both divergent ssoII promoters are located in the 109-bp intergenic spacer (2). Such an assumption, however, created a problem, since it was not clear how the methyltransferase interaction with the operator could stimulate transcription of ssoII.R, an essential step during establishment of a plasmid containing the ssoII genes in a naive host. In fact, bioinformatically predicted consensus promoter elements of the putative ssoII.R promoter overlapped with the M.SsoII binding site (2). Such an arrangement is expected to repress rather than activate ssoII.R transcription by bound M.SsoII, in the same way as in the case of ssoII.M transcription.
We felt that transcription regulation of ssoII genes needs to be revisited. For historical reasons, we studied transcription regulation in the Ecl18kI system. However, since the intergenic regions and adjacent DNA sequences in both Ecl18kI and SsoII are identical, there is no doubt that transcription regulation in both systems is identical too. The results reported here reveal that only the methyltransferase promoter was predicted correctly in the previous work. The restriction endonuclease promoter is located outside of the intergenic region, inside the coding sequence of the methyltransferase gene. Methyltransferase binding to the operator has little or no direct effect on transcription from the restriction endonuclease promoter, which becomes activated due to decreased competition from the methyltransferase promoter. The competition is caused by two effects: first, RNAP bound to the methyltransferase promoter decreases, but does not prevent, RNAP interaction with the restriction endonuclease promoter. Second, at conditions when both promoters are occupied by RNAP, transcription initiation from methyltransferase promoter occurs faster than from restriction endonuclease promoter.
The plasmid vectors used were pUC18/19 (8), pQE30 (9,10), and pFD51 (11). The entire Ecl18kI system was cloned into the polylinker of pUC19. The resultant plasmid was named pBcn(m + r + ). Plasmid pBcn(m + r -) was obtained from the pBcn(m + r + ) by digestion with BglII (cuts once within the ecl18kI.R gene), followed by fill-in with the Klenow enzyme and religation. Plasmid pBcn(mr -) was prepared by excision of an EcoRV fragment containing part of the ecl18kI.M gene from pBcn(m + r -).
Plasmid pRmGalK was prepared by cloning a fragment of ecl18kI DNA containing the entire Ecl18kI intergenic region and beginnings of Ecl18kI structural genes into promoterless plasmid pFD51; in pRmGalK the galK gene of pFD51 is controlled by ecl18kI.R promoter (Pr); the ecl18kI.M promoter (Pm) is also present and initiates transcription in the opposite direction. In plasmid pMrGalK, the galK gene of pFD51 is controlled by Pm; Pr is also present and initiates transcription in the opposite direction. In pResBSGalK, the galK gene of pFD51 is controlled by Pr; Pm is absent.
Plasmid p18Km(m + r -) was obtained from the p18Km plasmid carrying the ecl18kI genes (1) by digestion with BglII (cuts once within the ecl18kI.R gene), followed by fill-in with the Klenow enzyme and religation. This plasmid is compatible with plasmids described above and was used as a source of Ecl18kI methyltransferase M.Ecl18kI.

Proteins
RNAP and N-terminally hexahistidine-tagged M.Ecl18kI were purified as described in Refs. 14 and 22, respectively.
Total RNA preparation and primer extension reactions RNA was purified from E. coli K802 harboring indicated plasmids using SV Total RNA Isolation System (Promega). Purified samples were treated with DNase I and then repurified using the same kit. Total RNA (3 mg) was reverse-transcribed with 200 U RevertAid M-MuLV Reverse Transcriptase (Fermentas) in the presence of 1 pmol of g-32 P-labeled primer. Primer extension reactions were carried out at 428C for 60 min and terminated by a 5-min incubation at 708C. Samples were extracted with chloroform, ethanol-precipitated, dissolved in formamide-containing loading buffer and loaded on 7% sequencing gels. As markers, the products of DNA sequencing reactions performed with fmol DNA Cycle Sequencing System (Promega) using ecl18kI DNA as template and the same primer were used. Reaction products were revealed by autoradiography. 5 0 RACE was performed exactly as described in refs. (12) and (13).

In vitro transcription reactions
As templates for in vitro transcription, a PCR-amplified 290-bp-long ecl18kI fragment containing both ecl18kI promoters amplified from plasmid pBcn(m + r + ) or a 295-bp-long fragment containing only the ecl18kI.R promoter and the Ecl18kI.M operator (amplified from plasmid pResBSGalK) were used. Transcription reactions were set in 14 ml and contained 50 ng of transcription templates and various amounts of E. coli RNAP s 70 holoenzyme in a buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, 50 mg BSA, 5% glycerol. After a 5-10-min incubation at 378C, the reactions were supplemented with 2 ml of nucleotide hot mix [2 mM ATP, GTP and CTP, 500 mM UTP and 0.5 mCi [a-32 P]-UTP (3000 Ci/mmol) and 20 mg heparin] and incubated for additional 10 min at the same temperature. In the order of additions experiments, the first protein was incubated with DNA for 10 min followed by the addition of the second protein and an additional 10-min incubation prior to the addition of the hot mix. Reactions were terminated by the addition of 15 ml of formamide-containing loading buffer and loaded on a 7% sequencing gels.

Footprinting
KMnO 4 probing and DNase I footprinting were conducted under conditions used for in vitro transcription using 32 P-labeled promoter fragments as described (14). G+A sequencing reactions were carried out as described (15). Samples were applied on 6% sequencing gels and reaction products revealed using autoradiography.

Transcription of ecl18kI genes in vivo
The entire Ecl18kI system was cloned into E. coli plasmid pUC19. The resultant plasmid was named pBcn(m + r + ). Escherichia coli cells harboring pBcn(m + r + ) grew well and restricted the growth of vir phage (data not shown), indicating that both ecl18kI genes are expressed. To measure the abundance of ecl18kI transcripts in the presence or in the absence of M, RT-PCR analysis of RNA prepared from cells harboring pBcn(m + r + ) or its two derivatives was performed. Plasmid pBcn(m + r -) has a frame-shift mutation in the beginning of the R gene and does not produce restriction endonuclease. Plasmid pBcn(mr -) has both ecl18kI genes disrupted (see 'Materials and Methods' section). After reverse transcription of total cellular RNA with R or M-specific primers, the amounts of reverse-transcribed DNA were determined by registering threshold cycles in RT-PCR reactions for each of the genes monitored. As a control, the amount of transcripts from ampicillin-resistance gene bla present in pBcn(m + r + ) and its derivatives was determined alongside determinations of the amounts of ecl18kI transcripts and found to be very similar or identical in all cases (threshold cycles of 7.5 AE 0.4). A typical result is presented in Figure 1 and can be summarized as follows. For M transcripts, threshold cycle values for RNA purified from cells harboring pBcn(m + r + ) and pBcn(m + r -) were similar (12.0 AE 0.3). Threshold cycle values for M transcripts from cells harboring pBcn(mr -) were 7.3 AE 0.5. Thus, the presence of M leads to $2 5 (32-fold) decrease in steady-state levels of M transcripts. For R transcripts, threshold cycle values for RNA purified from cells harboring pBcn(m + r + ) and pBcn(m + r -) were 10.4 AE 0.4. The value was 14.4 AE 0.2 for RNA from pBcn(mr -)-harboring cells. Comparable threshold cycle values for pBcn(m + r + ) and pBcn(m + r -) exclude the effect of transcription polarity as the cause of decreased amounts of R transcripts in cells harboring pBcn(mr -). Thus, the presence of M leads to $2 4 (16-fold) increase in steady-state levels of R transcripts.
The reverse-transcription stage of the RT-PCR experiment was carried in the presence of large excess of genespecific primers. Therefore, some conclusions about the relative abundance of individual ecl18kI transcripts can be made. It appears that during stable maintenance of a plasmid containing functional Ecl18kI system (a condition of our experiment) the amount of R transcripts exceeds the amount of M transcripts (threshold cycle values of $10 and $12, correspondingly), though not by a large margin. Since the RT-PCR experiment measures the steady-state levels of transcripts, which depend both on the rate of transcript synthesis and its decay, no firm conclusions about transcription activity of ecl18kI promoters can be made.
Transcription initiation start points for ecl18kI genes were next established. Primer extension analysis of RNA prepared from cells harboring pBcn(m + r + ) with several ecl18kI specific primers revealed no primer extension products, possibly due to low levels of ecl18kI transcripts in the cell. The ecl18kI intergenic region and its flanking sequences were cloned, in both orientations, in front of promoterless galK gene of the plasmid pFD51. The resulting plasmids were introduced in galK À E. coli cells with or without a compatible plasmid that harbored intact M gene and its upstream sequences but lacked the R gene. Cells harboring a pFD51 derivative in which galK was under the control of Pm formed red colonies on McConkey agar plates, but only in the absence of compatible plasmid producing the methyltransferase (data not shown). RNA purified from these cells was subjected to primer extension analysis using a primer specific for the galK gene. A single, major, primer extension product was revealed in the absence of M (Figure 2A, lane 1). As can be seen from Figure 3, the position corresponding to primer extension product endpoint was preceded by an appropriately positioned À10 element cataat (consensus sequence TATAAT) and, 17 bases upstream, a À35 element ttgaaa (consensus sequence TTGACA). The two elements matched those bioinformatically predicted for SsoII promoter Pm in the earlier study (2). In addition to the major primer extension product, a series of longer primer extension products extending as much as 12 nt upstream of the major product with 1-nt increments were observed. The abundance of these products decreased for longer products. The initial transcribed sequence of Pm contains a run of four adenosines in positions +2 to +5. While this was not rigorously investigated, the longer primer extension products most likely result from transcript slippage during transcription through the run of adenosines. Transcript slippage is known to occur on some promoters containing runs of three or more identical nucleotides immediately downstream of the transcription start point and is a subject of biological regulation in some systems [see (16) and references therein]. Transcript slippage was also observed during transcription initiation in the EcoRV R-M system (17). The significance of transcript slippage, if any, for regulation of methyltransferase synthesis in the case of Ecl18kI remains to be determined. No primer extension product was detected when RNA from cells containing M encoded by a separate plasmid was analyzed (   upstream of the R gene initiation codon (Figure 3). These products were undetectable in the absence of M ( Figure 2B, lane 1). Upstream of primer extension product end points there is an appropriately positioned sequence tattat with similarity to consensus À10 promoter element sequence TATAAT (Figure 3). However, a sequence similar to the À35 promoter element consensus is absent. The 5 0 ends of RNAs corresponding to primer extension products are located upstream of the start point of bioinformatically predicted restriction endonuclease promoter of SsoII (2) and overlap with the translation start codon of the Ecl18kI methyltransferase gene. No primer extension products corresponding to transcripts initiated from previously predicted SsoII Pr (2) could be detected (data not shown).
We used 5 0 -RACE as an alternative approach to confirm Pr start point(s). A single TAP-stimulated PCR amplification product was revealed during the analysis of RNA purified from cells containing pBcn(m + r + ) (data not shown). Upon sequencing, sequences corresponding to three start points, two matching those revealed by primer extension (above), and another one located 112 bp upstream of the restriction endonuclease gene initiating codon, were found (see Figure 3). We conclude that primer extension experiments indeed reveal a promoter that is distinct from the one predicted in the earlier work on the SsoII system (2). Conversely, the predicted endonuclease promoter either does not exist or is inactive in our conditions. The fact that the Pr is located outside of the ecl18kI intergenic region indicates that the mechanism of ecl18kI genes regulation must be distinct from that proposed previously.

Transcription of ecl18kI genes in vitro
Transcription from ecl18kI promoters was next investigated in vitro. A fragment of ecl18kI DNA containing both promoters was combined with E. coli RNAP s 70 holoenzyme in the presence or in the absence of purified His-tagged methyltransferase, and a single round of transcription was allowed by the addition of NTPs and heparin. The results revealed a single transcription product in the absence of M ( Figure 4A, lane 1). By primer extension, this product corresponded to Pm-initiated transcript (data not shown). In the presence of M, added either before or after RNAP, the abundance of this transcript decreased and another, longer, transcript became prominent ( Figure 4A, lanes 2 and 3). The start of this transcript was also mapped and found to match Pr transcription start points (data not shown). Thus, M decreases transcription from Pm and activates transcription from Pr in vitro. The results presented above do not allow one to determine whether transcription from Pr is activated directly (for example through a protein-protein contact between operator-bound M and RNAP), or activation is indirect, i.e. is a simple consequence of removing an RNAP molecule bound to Pm. To address this question, in vitro transcription and KMnO 4 probing on a mutant fragment that contained Pr and the M operator but lacked Pm, which was destroyed by a mutation that removed its À35 element, were performed. The results of the in vitro transcription experiment revealed robust activity from Pr that was unaffected by M ( Figure 5A, compare lanes 1 and 2). No transcription from Pm was evident, as expected. KMnO 4 probing revealed that Pr open complexes were formed on the mutant template both in the presence and in the absence of M ( Figure 5B, lanes 7-9). No open complex formation on Pm was observed, as expected. As judged by DNase I footprinting, M bound the mutant template normally, and protected DNA from position +15 to +67 with respect to the Pr transcription start point ( Figure 5B, lane 2).
The absence of direct effect of M on transcription from Pm could also be demonstrated in vivo ( Figure 5C). Primer extension analysis of RNA initiated from plasmid-borne Pr-galK fusion plasmid with disrupted Pm was performed. As can be seen, there was no increase in abundance of Pr-initiated transcripts when M was provided from a compatible plasmid (in fact, a small decrease was observed, which may have been due to interference of bound M with transcription initiated from Pr). From these experiments, we conclude that Pr is a strong promoter whose activity is masked by RNAP binding to an even stronger Pm. Clearly, the Ecl18kI methyltransferase activates Pr indirectly, by preventing promoter complex formation on Pm.
In vitro experiments presented above were conducted at conditions when RNAP was present in equimolar amounts with DNA fragments containing ecl18kI promoters. The results of these experiments suggest that formation of the Pr and Pm promoter complexes is mutually exclusive. However, experiments conducted in the presence of higher concentrations of RNAP suggest that this is not necessarily so. As can be seen from Figure 6A Figure 6A, lane 4). The effect appeared to be cooperative, since in the presence of intermediate concentrations of RNAP no gradual increase of Pr complexes was detected. We therefore conclude that both ecl18kI promoters can be simultaneously occupied by RNAP. The conclusion was also supported by the results of gelretardation experiments (data not shown) and DNase I footprinting conducted at two different concentrations of RNAP ( Figure 6B). As can be seen from the figure, at high RNAP concentrations, complexes at both promoters were formed ( Figure 6B, lane 3 0 ). In the presence of high concentrations of RNAP, the addition of M decreased open complex formation on Pm, as expected, but did not lead to further stimulation of complex formation Pr ( Figure 6A, lane 5).
Surprisingly, the results of single-round transcription did not follow the results of KMnO 4 probing. As can be seen from Figure 6C, increasing concentrations of RNAP led only to a marginal increase in the amounts of Pr transcripts in absence of M the (compare lanes 1 and 4). In contrast and as expected, the addition of M strongly increased the amount of Pr transcripts (and decreased the amount of Pm transcripts, lane 5). Thus, in the presence of excess RNAP, promoter complexes on Pr form in the absence of M but no full-sized transcripts from these complexes is produced.
To determine if promoter complexes formed on Pr in the absence of M are transcriptionally active, abortive transcription initiation reactions were performed. To reveal transcription initiation from Pr, the synthesis of CpApU from the CpA primer and radioactive UTP was monitored; to reveal transcription from Pm, the synthesis of ApUpC from the ApU primer and radioactive CTP was followed. The reactions contained enough RNAP to allow promoter complex formation on both promoters in the absence of M. In addition to the wild-type ecl18kI template containing both promoters and extending from À60 to +132 relative to Pr start point (-81 to +110 relative to Pm start point) templates containing mutations disrupting either Pr (-81 to +36 relative to Pm start point, +17 to +132 relative to Pr start point) or Pm (-60 to +53 relative to Pr start point, À2 to +111 relative to Pm start point) were also used as controls. As can be seen from Figure 6D, on template containing both promoters (R + O + M + ), both abortive trinucleotide diphosphates were synthesized by RNAP alone (Figure 6D, lanes 3  and 9). In the presence of M, the synthesis of ApUpC was strongly inhibited (compare lanes 3 and 4), while the CpApU synthesis was unaffected (compare lanes 9 and 10). Reactions with mutant templates (labeled  Figure 6D) confirmed that each trinucleotide was synthesized from the expected promoter since disruption of Pr abolished the synthesis of CpApU but not ApUpC (compare lanes 11 and 5), while disruption of Pm abolished the synthesis of ApUpC but not of CpApU (compare lanes 1 and 7). Thus, when RNAP is in excess, active promoter complexes on both ecl18kI promoters form in the absence of M, but full-sized transcripts from Pm only are produced.
An arrangement of promoters similar to that found in Ecl18kI has been described in immunity regions of several lambdoid phages (18). There, preferential transcription from one promoter was explained by a 'sitting duck' model, which posits that promoter from which RNAP escapes faster 'wins' over a promoter from which the escape is slow (19). We determined the time course of RNAP escape from Pr and Pm in the absence of the interfering promoter (Pm or Pr, respectively). The results are shown in Figure 7. As can be seen, in a single-round transcription experiment, the Pm transcript accumulated considerably faster than the Pr transcript (for example, at 60-s time point, no full-sized Pr transcript was produced, while substantial amounts of Pm transcript was already synthesized, compare lane 2 0 with lane 2). We propose that, in accordance with the 'sitting duck' model, faster promoter clearance from Pm leads to disruption of open complexes on Pr, which require more time to escape to elongation. As a result, more Pm transcripts are produced even when RNAP forms complexes on both Ecl18kI promoters.

DISCUSSION
In every type II R-M system studied today, special mechanisms exist that ensure coordinated expression of restriction endonuclease and methyltransferase genes.  . The time course of accumulation of full-sized ecl18kI transcripts in vitro. Escherichia coli RNAP s 70 holoenzyme was combined with DNA fragments containing the indicated promoters and a single round of transcription was initiated by the addition of NTPs and heparin. PCR fragments used as templates for in vitro transcription allowed the generation of similarly sized full-sized transcripts from both ecl18kI promoters. Reaction aliquots were withdrawn and transcription terminated at times indicated. Reaction products were resolved by denaturing PAGE and revealed by autoradiography.
The functional requirements for such regulation are 2-fold. First, during establishment of an R-M system in a naive host, methyltransferase needs to be expressed first and in amounts sufficient for complete modification of recognition sites in the host genome before restriction endonuclease activity appears. Second, during stable maintenance of an R-M system, expression of methyltransferase needs to be decreased to prevent inadvertent methylation (and therefore protection) of incoming foreign DNA, for example phage DNA. Regulation of R-M genes expression at the level of transcription appears to be the most economical from the point of view of expenditure of cellular resources.
Genetic architecture of various R-M systems imposes important constraints on molecular mechanisms that ensure coordinated gene expression. R-M system Ecl18kI studied in this work is characterized by divergent orientation of structural genes and a presence of a predicted helix-turn-helix domain in the N-terminal portion of methyltransferase. The intergenic region separating the two structural genes contains a palindromic sequence (an operator) to which the N-terminal domain of methyltransferase can bind to. Methyltransferase interaction with the operator provides a negative-feedback loop that autogeneously decreases transcription of the M gene and a positive-feedback loop that increases transcription of the R gene. R-M systems MspI (20) and EcoRII (21) are organized similarly to SsoII. In the case of MspI, transcription start points have been mapped (20) and it was shown that (i) the M gene transcript start point overlaps with the methylase binding site, suggesting that methylase inhibits M gene transcription by steric exclusion and (ii) the R gene transcript start point is located $50 bp downstream of the methylase binding site, suggesting direct activation of R gene transcription by the bound methylase.
The SsoII transcription start points have not been mapped. While it was a priori clear that negative regulation of the SsoII M gene transcription occurred most likely at the level of steric occlusion of the promoter by operator-bound methylase, the mechanism of activation of SsoII Pr remained less clear, especially since this promoter was predicted to be very close to Pm. The principal result of our work is the demonstration that Pr is located outside the intergenic region, in the beginning of the M gene. As a result, the two promoters are facing each other, their transcriptions start points separated by 50 bp of DNA. The Pr promoter identified here could not have been revealed by bioinformatic analysis alone due to its unusual location (outside the ecl18kI intergenic region and within the M gene) and low similarity to existing bioinformatic models of s 70 holoenzyme promoters. In fact, Pr should be considered a novel promoter as it lacks clear similarity to either À10/-35 or extended À10 promoters and yet, in the absence of interference from Pm, it acts as a strong promoter both in vitro and in vivo. The result underscores the fact that there may be a large number of promoters and RNAP-binding sites in bacterial genomes that escape identification by bioinformatic means and yet perform important functions. Further, it appears that molecular mechanisms of regulation of restriction endonuclease genes in Ecl18kI (SsoII) and MspI R-M systems are quite different despite the common architecture of these systems.
Our results suggest that at low RNAP concentrations, RNAP binding to Pm decreases the interaction with Pr, probably due to direct interference between front ends of RNAP molecules bound to either one of the two promoters. The binding of Ecl18kI methyltransferase directly interferes with promoter complex formation on Pm but per se has no effect on RNAP interaction with Pr. However, decreased complex formation on Pm allows the Pr complex to form more efficiently. Apparently, transcription from Pr proceeds through the methyltrasferaseoperator complex. The fate of this complex during the passage of Pr-initiated transcription elongation complex is unknown. It is attractive to speculate that such transcription dislodges methyltransferase from the complex, thus allowing promoter competition to be reenacted after each transcription initiation event from Pr, and thus providing a mechanism to fine-tune the amount of Pm and Pr transcripts in the cell. Pr is located more than 100 bp away from the R gene starting codon. It is possible that this long leader contributes to the regulation of Ecl18kI gene expression since Ecl18kI mRNAs may hybridize to one another over the full distance between their respective start sites. Such hybridization would block the M translation initiation region, but not the R initiation region, which may help in the transition from 'methylate only' stage when a plasmid containing Ecl18kI genes establishes in naive host to 'begin restriction' stage during stable maintenance of the plasmid.
At high RNAP concentrations, open complexes on both ecl18kI promoters form even in the absence of M. However, no full-sized Pr transcripts are produced. A likely explanation is that in the presence of transcription substrates, promoter complexes on Pr are disrupted by transcription elongation complexes initiated from Pm, which is a faster-clearing promoter. Both mechanisms of Pr activation by M (through increase of Pr open complex formation and through increase of productive transcription from Pr open complexes) may be operational in vivo and controlled not only by RNAP availability but also by DNA topology. The relative contribution of both mechanisms requires further investigation and is a subject of ongoing work in our laboratories.
There is an interesting question about the relationship between the HTH regulatory DNA-binding domains of methyltransferases such as M.Ecl18kI, M.MspI and M.EcoRII and C-proteins, small DNA-binding proteins that regulate transcription in many type II R-M systems (22). All C-proteins are evolutionarily related to each other and are also distant relatives of phage regulators such as the repressor. Recently, a comprehensive bioinformatics survey identified several hundred C-proteins that could be clustered into 10 families (23). In all cases that have been studied, C-proteins recognize duplicated palindromic binding sites called C-boxes. Binding of a C-protein dimer to the high-affinity site activates the R gene promoter. Binding to the low-affinity site represses it. Sequence analysis indicates that the HTH domain of M.Ecl18kI is distinct from C-proteins, that is, even most distant C-proteins are more closely related to each other than to M.Ecl18kI DNA-binding domain. Likewise, the structure of M.Ecl18kI operator is distinct from any known or predicted C-box. The operator contains a single rather than a duplicated palindrome and the inverted repeat in the operator is much longer than those in C-boxes. Our recent attempts to observe operator binding with recombinant HTH domain of M.Ecl18kI were unsuccessful (24), indicating that sequences in the methyltrasnferase part of M.Ecl18kI are required for DNA binding (the reverse is not true since M.Ecl18kI lacking the HTH domain is fully capable of site methylation). Structural characterization of M.Ecl18kI, alone and in complex with the operator, will be necessary to clarify these issues.