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• W1973152779 abstract "Although the archaeal transcription apparatus resembles the eukaryal RNA polymerase II system, many bacterial-like regulators can be found in archaea. Particularly, all archaeal genomes sequenced to date contain genes encoding homologues of Lrp (leucine-responsiveregulatory protein). Whereas Lrp-like proteins in bacteria are involved in regulation of amino acid metabolism, their physiological role in archaea is unknown. Although several archaeal Lrp-like proteins have been characterized recently, no target genes apart from their own coding genes have been discovered yet, and no ligands for these regulators have been identified so far. In this study, we show that the Lrp-like protein LysM from Sulfolobus solfataricus is involved in the regulation of lysine and possibly also arginine biosynthesis, encoded by the lys gene cluster. Exogenous lysine is the regulatory signal for lysgene expression and specifically serves as a ligand for LysM by altering its DNA binding affinity. LysM binds directly upstream of the TFB-responsive element of the intrinsically weak lysWpromoter, and DNA binding is favored in the absence of lysine, whenlysWXJK transcription is maximal. The combined in vivo and in vitro data are most compatible with a model in which the bacterial-like LysM activates the eukarya-like transcriptional machinery. As with transcriptional activation by Escherichia coli Lrp, activation by LysM is apparently dependent on a co-activator, which remains to be identified. Although the archaeal transcription apparatus resembles the eukaryal RNA polymerase II system, many bacterial-like regulators can be found in archaea. Particularly, all archaeal genomes sequenced to date contain genes encoding homologues of Lrp (leucine-responsiveregulatory protein). Whereas Lrp-like proteins in bacteria are involved in regulation of amino acid metabolism, their physiological role in archaea is unknown. Although several archaeal Lrp-like proteins have been characterized recently, no target genes apart from their own coding genes have been discovered yet, and no ligands for these regulators have been identified so far. In this study, we show that the Lrp-like protein LysM from Sulfolobus solfataricus is involved in the regulation of lysine and possibly also arginine biosynthesis, encoded by the lys gene cluster. Exogenous lysine is the regulatory signal for lysgene expression and specifically serves as a ligand for LysM by altering its DNA binding affinity. LysM binds directly upstream of the TFB-responsive element of the intrinsically weak lysWpromoter, and DNA binding is favored in the absence of lysine, whenlysWXJK transcription is maximal. The combined in vivo and in vitro data are most compatible with a model in which the bacterial-like LysM activates the eukarya-like transcriptional machinery. As with transcriptional activation by Escherichia coli Lrp, activation by LysM is apparently dependent on a co-activator, which remains to be identified. RNA polymerase TATA-binding protein transcription factor TFB-responsive element avian myeloblastosis virus reverse transcriptase α-aminoadipic acid electrophoretic mobility shift assay preinitiation complex Since the discovery of archaea as a distinct domain of life, many studies have focused on archaeal transcription. It has become clear that although archaea resemble bacteria with respect to their cellular and genetic organization, their transcriptional apparatus is fundamentally different from that of bacteria. Their RNA polymerase (RNAP)1 is much more related to the eukaryal RNAPII system regarding subunit complexity and sequence homology (1Langer D. Hain J. Thuriaux P. Zillig W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5768-5772Crossref PubMed Scopus (262) Google Scholar). Thus, archaeal RNAP consists of at least 10 subunits in contrast to the five-subunit bacterial RNAP core enzyme. As in eukarya, archaeal transcription initiation is preceded by the binding of the TATA-binding protein (TBP) to a TATA-like sequence called the TATA-box and subsequent binding of transcription factor B (TFB). Archaeal TBP and TFB are highly homologous to the eukaryal TBP and TFIIB, respectively. However, archaeal TBP is not complexed with TBP-associated factors as in eukarya (2Qureshi S.A. Bell S.D. Jackson S.P. EMBO J. 1997; 16: 2927-2936Crossref PubMed Scopus (120) Google Scholar), and there is no evidence that archaeal genomes encode TBP-associated factor homologues. The archaeal TATA-box is 8 bp in length and is located ∼25 bp upstream of the start of transcription. Directly upstream of the TATA-box, a purine-rich sequence is present, called the TFB-responsive element (BRE). The BRE was shown to be an important determinant in directionality of transcription and promoter strength through interaction with a C-terminal helix-turn-helix domain of TFB (3Bell S.D. Kosa P.L. Sigler P.B. Jackson S.P. Proc. Natl. Acad. Sci. 1999; 96: 13662-13667Crossref PubMed Scopus (128) Google Scholar, 4Littlefield O. Korkhin Y. Sigler P.B. Proc. Natl. Acad. Sci. 1999; 96: 13668-13673Crossref PubMed Scopus (134) Google Scholar). The TF(II)B-BRE interaction is a conserved feature between archaea and eukarya. Once TBP and TFB are bound to the promoter, RNAP is recruited, involving an interaction between the RpoK subunit of RNAP and the N-terminal zinc ribbon domain of TFB (5Magill C.P. Jackson S.P. Bell S.D. J. Biol. Chem. 2001; 276: 46693-46696Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar).Although no archaeal homologues of eukaryal TFIIA, TFIIF, and TFIIH have been identified, a protein homologous to the N-terminal region of the α-subunit of eukaryal TFIIE is present in archaea. This archaeal TFE stimulates transcription from promoters with suboptimal TATA-box sequences or in cases where TBP is limiting (6Bell S.D. Brinkman A.B. van der Oost J. Jackson S.P. EMBO Rep. 2001; 2: 133-138Crossref PubMed Scopus (80) Google Scholar, 7Hanzelka B.L. Darcy T.J. Reeve J.N. J. Bacteriol. 2001; 183: 1813-1818Crossref PubMed Scopus (66) Google Scholar). Whereas eukaryal TFIIE is strictly necessary for transcription, archaeal TFE appears to be dispensable for basal transcription in vitro, although it may play a stimulatory role in transcription initiation at specific promoters.Although the basal components of the archaeal and eukaryal transcription machineries are very similar, regulatory proteins do not appear to be conserved between the two domains. Instead, archaeal genomes contain many regulators previously identified only in bacteria, so-called bacterial-archaeal regulators (8Bell S.D. Jackson S.P. Curr. Opin. Microbiol. 2001; 4: 208-213Crossref PubMed Scopus (172) Google Scholar). In particular, homologues of the Lrp/AsnC family of regulators appear to be widely distributed among both bacteria and archaea. Several bacterial as well as archaeal genomes contain up to 10 Lrp-like paralogues. Escherichia coli Lrp (leucine-responsive regulatoryprotein) is the paradigm that has been studied extensively (9Calvo J.M. Matthews R.G. Microbiol. Rev. 1994; 58: 466-490Crossref PubMed Google Scholar, 10Newman E.B. Lin R. Annu. Rev. Microbiol. 1995; 49: 747-775Crossref PubMed Scopus (140) Google Scholar). It is a global regulator controlling the expression of up to 75 genes (11Lin R. D'Ari R. Newman E.B. J. Bacteriol. 1992; 174: 1948-1955Crossref PubMed Google Scholar, 12Ernsting B.R. Atkinson M.R. Ninfa A.J. Matthews R.G. J. Bacteriol. 1992; 174: 1109-1118Crossref PubMed Google Scholar). E. coli Lrp either represses or activates transcription, the effect of which is sometimes modulated by leucine. The target genes of E. coli Lrp encode enzymes that are directly or indirectly related to amino acid metabolism. This also appears to be the case for several specific (nonglobal) bacterial Lrp-like regulators from different bacteria. In archaea, the exact role of the numerous Lrp-like proteins has not been established. Several archaeal Lrp-like proteins have been characterized recently (13Napoli A. van der Oost J. Sensen C.W. Charlebois R.L. Rossi M. Ciaramella M. J. Bacteriol. 1999; 181: 1474-1480Crossref PubMed Google Scholar, 14Bell S.D. Jackson S.P. J. Biol. Chem. 2000; 275: 31624-31629Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Brinkman A.B. Dahlke I. Tuininga J.E. Lammers T. Dumay V. de Heus E. Lebbink J.H. Thomm M. de Vos W.M. van der Oost J. J. Biol. Chem. 2000; 275: 38160-38169Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 16Enoru-Eta J. Gigot D. Thia-Toong T.L. Glansdorff N. Charlier D. J. Bacteriol. 2000; 182: 3661-3672Crossref PubMed Scopus (49) Google Scholar). For two of these proteins, Lrs14 from Sulfolobus solfataricus and LrpA from Pyrococcus furiosus, anin vitro regulatory function could be assigned; both showed negative autoregulation independent of any amino acid ligand (14Bell S.D. Jackson S.P. J. Biol. Chem. 2000; 275: 31624-31629Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Brinkman A.B. Dahlke I. Tuininga J.E. Lammers T. Dumay V. de Heus E. Lebbink J.H. Thomm M. de Vos W.M. van der Oost J. J. Biol. Chem. 2000; 275: 38160-38169Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Moreover, the three-dimensional structure of P. furiosusLrpA was determined, providing the structural basis for understanding LrpA-DNA as well as LrpA-ligand interactions (17Leonard P.M. Smits S.H.J. Sedelnikova S.E. Brinkman A.B. de Vos W.M. van der Oost J. Rice D.W. Rafferty J.B. EMBO J. 2000; 20: 990-997Crossref Scopus (124) Google Scholar). However, neither the identity of this ligand nor the role of archaeal Lrp-like proteins in the expression of other genes has been determined.To provide a suitable model system for analyzing the function of Lrp-like proteins in archaea, we have screened the genome of the hyperthermophilic archaeon S. solfataricus for the presence of Lrp-like proteins whose function, target, and ligand may be readily predicted. We have identified and characterized the Lrp-like protein LysM, the gene of which is clustered with genes encoding lysine biosynthetic enzymes. We show here that expression of thelysWXJK genes is regulated by the presence of lysine in the medium. In vitro LysM binds to the lysW promoter, and binding is favored in the absence of lysine, when lysgene expression is maximal. A model is proposed for lysine-modulated activation of transcription through LysM, for the first time indicating that a bacterial-like regulator may activate the eukarya-like archaeal transcriptional machinery. It appears that Lrp-like proteins are functionally equivalent in the bacterial and archaeal domains, despite the fundamental differences in transcriptional machineries.DISCUSSIONGenomic sequence data have revealed that Lrp-like proteins are ubiquitously present in bacteria and archaea. Although Lrp fromE. coli is the archetype Lrp-like protein, more than a dozen bacterial Lrp-like proteins have been studied either in vitro or using genetic approaches, and it is clear that these proteins are all involved in the regulation of amino acid metabolism and that amino acids serve as ligands for Lrp-like proteins.2 Efficient genetic tools to study gene regulation (like recombination or gene disruption) are not yet available for hyperthermophilic archaea, and we have used a bioinformatics approach in combination with in vivo and in vitro analyses to identify and study the archaeal Lrp-like protein LysM from S. solfataricus, the gene of which is present in the lysgene cluster. This allowed us for the first time to study the role and function of an archaeal Lrp-like protein in relation to its physiological target genes.Regulation of lys transcription in S. solfataricus is responsive to lysine only, which strongly suggests that the lys genes are involved in lysine biosynthesis, most likely via a pathway similar to that of T. thermophilus, where gene disruption of the homologous lys cluster resulted in lysine auxotrophy (24Nishida H. Nishiyama M. Kobashi N. Kosuge T. Hoshino T. Yamane H. Genome Res. 1999; 9: 1175-1183Crossref PubMed Scopus (105) Google Scholar). The pathway utilized here involves AAA rather than diaminopimelic acid, the typical bacterial precursor (25Kosuge T. Hoshino T. FEMS Microbiol. Lett. 1998; 169: 361-367PubMed Google Scholar, 26Kobashi N. Nishiyama M. Tanokura M. J. Bacteriol. 1999; 181: 1713-1718Crossref PubMed Google Scholar). Although arginine has no effect on lystranscription, we cannot rule out the possibility thatlys-encoded enzymes are also functional in arginine biosynthesis, as was proposed for the lys gene cluster ofPyrococcus horikoshii (24Nishida H. Nishiyama M. Kobashi N. Kosuge T. Hoshino T. Yamane H. Genome Res. 1999; 9: 1175-1183Crossref PubMed Scopus (105) Google Scholar). Moreover, dual activity has been measured for LysJ and LysK of T. thermophilus, homologues ofS. solfataricus LysJ and LysK, respectively, suggesting that the lys gene cluster could indeed be involved in arginine as well as lysine biosynthesis (23Miyazaki J. Kobashi N. Nishiyama M. Yamane H. J. Bacteriol. 2001; 183: 5067-5073Crossref PubMed Scopus (40) Google Scholar, 33Miyazaki J. Kobashi N. Fujii T. Nishiyama M. Yamane H. FEBS Lett. 2002; 512: 269-274Crossref PubMed Scopus (27) Google Scholar).Why is the lys gene cluster of S. solfataricusorganized in a constitutive (lysYZM) and a regulated part (lysWXJK)? Possibly, down-regulation of thelysYZM is not permitted because this would abolish regulation of the lysWXJK genes through LysM, the gene of which is co-transcribed with lysZY. Our data do not exclude the possibility that LysM has additional targets in the genome ofS. solfataricus, but if this were the case, down-regulation of lysM could result in an even more serious loss of regulatory capacity in S. solfataricus. Alternatively, one of the enzyme activities encoded by lysYZM could be indispensable for growth under the conditions tested. The genomic organization of lysM in the lys cluster is identical in other Sulfolobus species and functionally comparable with that of A. pernix, where thelysYZM cluster is inverted (Fig. 1A), most likely allowing a similar mode of regulation.Although the role of lysW and lysX has not been demonstrated experimentally, we speculate that they are specific for the prokaryotic AAA lysine biosynthesis route proposed by Nishidaet al. (24Nishida H. Nishiyama M. Kobashi N. Kosuge T. Hoshino T. Yamane H. Genome Res. 1999; 9: 1175-1183Crossref PubMed Scopus (105) Google Scholar), since they are clustered within thelys clusters of T. thermophilus, S. acidocaldarius, S. tokodai, A. pernix, Pyrococcus species, and Ferroplasma acidarmanus and because the classical arginine or lysine pathways do not involve such genes. LysX is 24% identical to RimK ofE. coli, which was shown to be a post-translational modification enzyme, catalyzing the coupling of four glutamate residues to the C terminus of the S6 ribosomal protein (34Kang W.K. Icho T. Isono S. Kitakawa M. Isono K. Mol. Gen. Genet. 1989; 217: 281-288Crossref PubMed Scopus (58) Google Scholar). However, we found that in several prokaryotic genomes rimK-like genes are clustered with amino acid biosynthesis genes (not shown), suggesting that the catalytic activity of the rimK-encoded protein here is not utilized for a post-translational modification but rather in amino acid biosynthesis. Interestingly, Galperin et al. (35Galperin M.Y. Koonin E.V. Protein Sci. 1997; 6: 2639-2643Crossref PubMed Scopus (239) Google Scholar) demonstrated that RimK belongs to a superfamily of enzymes with a so-called “ATP grasp” fold. This family includes enzymes like d-alanine-d-alanine ligase, glutathione synthetase, biotin carboxylase, and carbamoyl phosphate synthetase. All of these enzymes possess ATP-dependent carboxylate-amine ligase activity (i.e. the capacity to form a peptide bond). In the proposed AAA-dependent lysine biosynthesis route described by Nishida et al. (24Nishida H. Nishiyama M. Kobashi N. Kosuge T. Hoshino T. Yamane H. Genome Res. 1999; 9: 1175-1183Crossref PubMed Scopus (105) Google Scholar), LysX was predicted to catalyze a similar reaction, namely connecting the amino group of AAA to the carboxyl group of a yet unidentified molecule. By doing so, LysX catalyzes a reaction functionally analogous to that of ArgA (N-acteylglutamate synthase) in the classical arginine biosynthesis pathway, the gene of which is absent in all lys clusters shown in Fig. 1A.The small protein encoded by lysW has no homologues in the data base, apart from lysW genes found in thelys clusters depicted in Fig. 1A. A PSI-BLAST analysis (36Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59182) Google Scholar) of LysW showed that this small protein is homologous to the N-terminal domain of archaeal TFB transcription factors (not shown). After four iterations, an expect value of ≥10−11was obtained with TFB N-terminal domains from several archaea. This domain consists of a zinc ribbon (37Zhu W. Zeng Q. Colangelo C.M. Lewis M. Summers M.F. Scott R.A. Nat. Struct. Biol. 1996; 3: 122-124Crossref PubMed Scopus (124) Google Scholar), and the two CPXCG “zinc knuckle” motifs that bind the zinc atom are well conserved in LysW. The zinc ribbon domain of Sulfolobus TFB has been shown to be involved in the recruitment of RNA polymerase (RNAP) (38Bell S.D. Jackson S.P. J. Biol. Chem. 2000; 275: 12934-12940Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), through interaction with the RpoK subunit of RNAP (5Magill C.P. Jackson S.P. Bell S.D. J. Biol. Chem. 2001; 276: 46693-46696Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Generally, zinc ribbon domains mediate protein-protein or protein-DNA interactions and can be found for instance in several eukaryal transcription factors. LysW may therefore interact with one of the encoded enzymes of thelys gene cluster, perhaps acting as a regulatory subunit for the respective enzymatic activity, or it could play a regulatory role in lys transcription. However, LysW is also encoded by thelys gene cluster of T. thermophilus, which possesses a bacterial basal transcription machinery, and it is questionable whether involvement in transcription here is possible. It should be noted, however, that the RpoK subunit, with which the TFB zinc ribbon interacts, is a conserved subunit in RNAPs of eukarya (RPB6) and bacteria (ω). Unfortunately, our attempts to produce the LysW protein in E. coli were unsuccessful.The data presented in this study strongly suggest that LysM acts as a transcriptional activator for PlysW. We found that transcription from PlysW is maximal when lysine is absent, and under these conditions the affinity of LysM for binding this promoter is the highest. Conversely, in the presence of lysine, transcription is lowest, and the binding affinity of LysM is decreased. It is most likely that LysM occupies PlysW preferably when lysine is absent, thereby somehow activating this promoter. Although lysine reduces rather than eliminates LysM-DNA binding, this reduction is expected to be physiologically important. As shown in Fig. 4, D and E, the effect of lysine is maximal at a low LysM concentration, presumably a relevant cellular condition (using Western blotting analysis, we roughly estimated the abundance of LysM to be about 0.01% of total soluble protein, not shown). This reduction rather than elimination of binding has also been observed for other (bacterial) Lrp-like proteins (39Ernsting B.R. Denninger J.W. Blumenthal R.M. Matthews R.G. J. Bacteriol. 1993; 175: 7160-7169Crossref PubMed Google Scholar, 40Roesch P.L. Blomfield I.C. Mol. Microbiol. 1998; 27: 751-761Crossref PubMed Scopus (66) Google Scholar, 41Zhi J. Mathew E. Freundlich M. Mol. Microbiol. 1999; 32: 29-40Crossref PubMed Scopus (26) Google Scholar, 42Madhusudhan K.T. Hester K.L. Friend V. Sokatch J.R. J. Bacteriol. 1997; 179: 1992-1997Crossref PubMed Google Scholar, 43Jafri S. Evoy S. Cho K. Craighead H.G. Winans S.C. J. Mol. Biol. 1999; 288: 811-824Crossref PubMed Scopus (38) Google Scholar) and may therefore be a general feature of Lrp-like proteins. Using in vivo formaldehyde cross-linking followed by immunoprecipitation of cross-linked LysM-DNA complexes, we have attempted to relate results from in vitrobinding studies with in vivo LysM promoter occupation, but unfortunately the results of these experiments were irreproducible. Nevertheless, to a certain extent, our study is comparable with the regulation of E. coli ilvIH by Lrp. For example, Lrp activates ilvIH transcription, and this activation is decreased when leucine is present in the medium (44Platko J.V. Willins D.A. Calvo J.M. J. Bacteriol. 1990; 172: 4563-4570Crossref PubMed Google Scholar). In accordance, the Lrp-ilvIH affinity in vitro is reduced but not eliminated by leucine (45Ricca E. Aker D.A. Calvo J.M. J. Bacteriol. 1989; 171: 1658-1664Crossref PubMed Google Scholar). For ilvIH, this reduction of binding in vitro could be related to an in vivodecrease in promoter occupancy using in vivo footprinting experiments (46Marasco R. Varcamonti M., La Cara F. Ricca E., De Felice M. Sacco M. J. Bacteriol. 1994; 176: 5197-5201Crossref PubMed Google Scholar), and we anticipate that this in vitro-in vivo relationship can also be made for LysM.Our DNase I footprint data support the possibility that LysM is an activator, since it showed that LysM protects the bases −46 to −59 relative to the transcriptional start site, whereas the TBP-TFB-RNAP preinitiation complex has previously been shown to protect the bases −43 to +8 at Sulfolobus viral T6 promoter (38Bell S.D. Jackson S.P. J. Biol. Chem. 2000; 275: 12934-12940Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Hence, LysM binding is not expected to interfere with or occupy the target sites for TBP, TFB, or RNAP but rather binds upstream of these proteins, as is usually the case for activators. In many cases, these activators recruit components or stabilize binding of the preinitiation complex (PIC) through direct contacts. However, such direct contacts have not yet been shown for Lrp-like proteins. Alternatively, activation could involve promoter remodeling in which the secondary structure of the promoter DNA is changed, for instance by bending the DNA in a certain angle. This altered DNA structure could subsequently be recognized more efficiently by one of the components of the PIC. In this case, activation of transcription would be independent of direct contacts between the activator and the PIC components. As shown in Fig.5A, binding of LysM induces several DNase I-hypersensitive sites, indicative of DNA secondary structure changes. Interestingly, one of the hypersensitive sites is located between the BRE and TATA-box, representing a structural alteration that is potentially able to alter the interaction properties of TBP/TFB with the DNA. In addition, the intensity of some of the LysM-induced hypersensitive sites is somewhat changed in the presence of lysine. However, the magnitude of this effect is less obvious than the effect of lysine observed in EMSAs (Fig. 4). We have tested the ability of LysM to bend its target DNA by using the pBEND2 system described by Kim et al. (47Kim J. Zwieb C., Wu, C. Adhya S. Gene (Amst.). 1989; 85: 15-23Crossref PubMed Scopus (321) Google Scholar). For this purpose, we cloned the 24-bp fragment used for Fig. 5, B and C, into pBEND2 and used it as described (47Kim J. Zwieb C., Wu, C. Adhya S. Gene (Amst.). 1989; 85: 15-23Crossref PubMed Scopus (321) Google Scholar), but no LysM-induced bending was observed, either in the presence or absence of lysine. The possibility cannot be ruled out, however, that low affinity binding to sequences outside the chosen sequence fragment contributes to the LysM-DNA interaction and bending.Our in vitro transcription experiments showed that compared with the T6 control promoter (2Qureshi S.A. Bell S.D. Jackson S.P. EMBO J. 1997; 16: 2927-2936Crossref PubMed Scopus (120) Google Scholar), transcription from PlysY and PlysW is very weak. We showed previously that PlysYcould be stimulated by the addition of TFE (6Bell S.D. Brinkman A.B. van der Oost J. Jackson S.P. EMBO Rep. 2001; 2: 133-138Crossref PubMed Scopus (80) Google Scholar) (referred to as PargC), but this was not possible for PlysW. Apparently, both promoters are intrinsically weak promoters, which is in agreement with the low homology to the Sulfolobusconsensus promoter sequence. It is therefore possible that binding of TBP and/or TFB might be impaired at PlysW. In agreement with this, in an EMSA we could not observe any interaction between PlysW DNA and (combinations of) these transcription factors, and the addition of LysM or lysine had no effect (not shown). Altogether, our results thus suggest that the intrinsic activity of PlysW promoters is very low. In contrast, lysWXJKmRNA could easily be detected in Northern blotting and primer extension experiments, suggesting efficient transcription in vivo. Since we have proven that both PlysY and PlysW are true promoters in vivo, we suggest that at least for PlysW, additional factors like co-regulators may be required for efficient transcription. Thus, under our experimental conditions, LysM is not able to activate transcription, but in the presence of such an unidentified factor, transcription may take place. In comparison, some E. coli promoters that belong to the Lrp regulon require an additional DNA-binding protein (e.g.integration host factor (IHF) (48Paul L. Blumenthal R.M. Matthews R.G. J. Bacteriol. 2001; 183: 3910-3918Crossref PubMed Scopus (24) Google Scholar), histone-like protein H-NS (49Levinthal M. Lejeune P. Danchin A. Mol. Gen. Genet. 1994; 242: 736-743Crossref PubMed Scopus (25) Google Scholar), or catabolite activator protein (CAP) (50Mathew E. Zhi J. Freundlich M. J. Bacteriol. 1996; 178: 7234-7240Crossref PubMed Google Scholar, 51Weyand N.J. Braaten B.A. van der Woude M. Tucker J. Low D.A. Mol. Microbiol. 2001; 39: 1504-1522Crossref PubMed Scopus (44) Google Scholar)). To identify such proteins in S. solfataricus, we have taken several approaches. First, in our in vitro transcription experiments, we have added cell extracts of S. solfataricus grown in the absence of lysine, but no stimulation of transcription in vitro was observed. Second, we have performed pull-down experiments in which glutathione S-transferase (GST) was fused to LysM and immobilized on glutathione-agarose beads. An S. solfataricuscell extract was subsequently screened for proteins interacting with GST-LysM. We found that a single protein interacted with LysM, but this protein was identified as the LysM protein itself, most likely being the result of multimerization of GST-LysM and wild-type LysM during the experiment (data not shown).The observation that LysM is conserved in the lysclusters of three Sulfolobus species and in A. pernix suggests that it plays a similar role in these organisms. We have therefore compared the sequence of their putativelysW promoters to identify a possible consensus LysM binding site. Indeed, a conserved GGTTC inverted repeat element is present, as shown in Fig. 8. For the putativelysW promoters of Sulfolobus species, the position of the presumptive LysM binding site is very similar (overlapping the lysM stop codon), whereas in A. pernix, where the lys gene cluster is organized in a somewhat different way (see Fig. 1A), the putative LysM site is centered between the lysY and lysW genes. It is remarkable that this LysM binding site is conserved and highly palindromic, since this is usually not the case for naturally occurring binding sites of Lrp-like proteins (9Calvo J.M. Matthews R.G. Microbiol. Rev. 1994; 58: 466-490Crossref PubMed Google Scholar, 15Brinkman A.B. Dahlke I. Tuininga J.E. Lammers T. Dumay V. de Heus E. Lebbink J.H. Thomm M. de Vos W.M. van der Oost J. J. Biol. Chem. 2000; 275: 38160-38169Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 52Madhusudhan K.T. Huang N. Sokatch J.R. J. Bacteriol. 1995; 177: 636-641Crossref PubMed Google Scholar). We have derived a consensus LysM site from the alignment given in Fig. 8, and we used this sequence to screen the S. solfataricus and A. pernix genomes for LysM sites, but no additional LysM binding sites could be identified.Several archaeal Lrp-like proteins have been characterized recently (13Napoli A. van der Oost J. Sensen C.W. Charlebois R.L. Rossi M. Ciaramella M. J. Bacteriol. 1999; 181: 1474-1480Crossref PubMed Google Scholar, 14Bell S.D. Jackson S.P. J. Biol. Chem. 2000; 275: 31624-31629Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Brinkman A.B. Dahlke I. Tuininga J.E. Lammers T. Dumay V. de Heus E. Lebbink J.H. Thomm M. de Vos W.M. van der Oost J. J. Biol. Chem. 2000; 275: 38160-38169Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 16Enoru-Eta J. Gigot D. Thia-Toong T.L. Glansdorff N. Charlier D. J. Bacteriol. 2000; 182: 3661-3672Crossref PubMed Scopus (49) Google Scholar), but LysM is the first for which a clear physiological role has been demonstrated. Unlike previously studied archaeal Lrp-like proteins, lysM is expressed constitutively and not negatively autoregulated. Moreover, LysM strongly resembles bacterial Lrp-like proteins and appears to be a specific rather than a global regulator, since it is clustered with its target genes. However, we cannot exclude the possibility that LysM has additional targets in theS. solfataricus genome, and experiments are necessary to confirm this. Furthermore, all bacterial Lrp-like proteins characterized to date act as transcriptional repressors or activators involved in the regulation of amino acid metabolism, and all ligands identified so far were found to be amino acids. In analogy, the p" @default.
• W1973152779 created "2016-06-24" @default.
• W1973152779 creator A5007026827 @default.
• W1973152779 creator A5010586842 @default.
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• W1973152779 date "2002-08-01" @default.
• W1973152779 modified "2023-10-03" @default.
• W1973152779 title "The Sulfolobus solfataricus Lrp-like Protein LysM Regulates Lysine Biosynthesis in Response to Lysine Availability" @default.
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