Effects of Inorganic Polyphosphate on the Proteolytic and DNA-binding Activities of Lon in Escherichia coli

Lon belongs to a unique group of proteases that bind to DNA and is involved in the regulation of several important cellular functions, including adaptation to nutritional downshift. Previously, we revealed that inorganic polyphosphate (polyP) increases in Escherichia coli in response to amino acid starvation and that it stimulates the degradation of free ribosomal proteins by Lon. In this work, we examined the effects of polyP on the proteolytic and DNA-binding activities of Lon. An order-of-addition experiment suggested that polyP first binds to Lon, which stimulates Lon-mediated degradation of ribosomal proteins. A polyP-binding assay using Lon deletion mutants showed that the polyP-binding site of Lon is localized in the ATPase domain. Because the same ATPase domain also contains the DNA-binding site, polyP can compete with DNA for binding to Lon. In fact, an equimolar amount of polyP almost completely inhibited DNA-Lon complex formation, suggesting that Lon binds to polyP with a higher affinity than it binds to DNA. Collectively, our results showed that polyP may control the cellular activity of Lon not only as a protease but also as a DNA-binding protein. Lon belongs to a unique group of proteases that bind to DNA and is involved in the regulation of several important cellular functions, including adaptation to nutritional downshift. Previously, we revealed that inorganic polyphosphate (polyP) increases in Escherichia coli in response to amino acid starvation and that it stimulates the degradation of free ribosomal proteins by Lon. In this work, we examined the effects of polyP on the proteolytic and DNA-binding activities of Lon. An order-of-addition experiment suggested that polyP first binds to Lon, which stimulates Lon-mediated degradation of ribosomal proteins. A polyP-binding assay using Lon deletion mutants showed that the polyP-binding site of Lon is localized in the ATPase domain. Because the same ATPase domain also contains the DNA-binding site, polyP can compete with DNA for binding to Lon. In fact, an equimolar amount of polyP almost completely inhibited DNA-Lon complex formation, suggesting that Lon binds to polyP with a higher affinity than it binds to DNA. Collectively, our results showed that polyP may control the cellular activity of Lon not only as a protease but also as a DNA-binding protein. Inorganic polyphosphate (polyP) 1The abbreviations used are: polyP, inorganic polyphosphate; MBP, maltose-binding protein. is a linear polymer of many hundreds of orthophosphate residues linked together by ATP-like high energy phosphoanhydride bonds. This polymer naturally occurs in microbes, fungi, plants, and animals (1Kornberg A. J. Bacteriol. 1995; 177: 491-496Crossref PubMed Scopus (474) Google Scholar). In Escherichia coli, the level of polyP is very low in the exponential phase, but it increases up to 1000-fold in response to amino acid starvation and during the onset of the stationary phase (2Rao N.N. Kornberg A. J. Bacteriol. 1996; 178: 1394-1400Crossref PubMed Google Scholar, 3Kuroda A. Murphy H. Cashel M. Kornberg A. J. Biol. Chem. 1997; 272: 21240-21243Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). We demonstrated previously that polyP forms a complex with the ATP-dependent Lon protease, an enzyme that degrades intracellular proteins during nutritional downshift (4Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (297) Google Scholar). We further showed that most of the proteins degraded by the polyP-Lon were free ribosomal proteins. Thus, it appears that polyP helps mediate adaptation to nutritional downshift by directing the degradation of ribosomal proteins. PolyP also binds to ribosomal proteins, suggesting that its interaction with the substrate may be important for the stimulation of degradation (4Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (297) Google Scholar). However, whether polyP first binds to Lon or to the ribosomal proteins has not been established. Lon is a highly conserved enzyme present in Archaea and eubacteria as well as in the mitochondria of eukaryotes. The Lon monomer includes an N terminus of unknown function, a central ATPase containing a typical ATP-binding motif, and a C-terminal proteolytic domain with a catalytically active residue at Ser-679 (5Chin D.T. Goff S.A. Webster T. Smith T. Goldberg A.L. J. Biol. Chem. 1988; 263: 11718-11728Abstract Full Text PDF PubMed Google Scholar, 6Maurizi M.R. Experimentia (Basel). 1992; 48: 178-201Crossref PubMed Scopus (312) Google Scholar, 7Yoshida M. Amano T. FEBS Lett. 1995; 359: 1-5Crossref PubMed Scopus (93) Google Scholar, 8Amerik A.Yu. Antonov V.K. Gorbalenya A.E. Kotova S.A. Rotanova T.V. Shimbarevich E.V. FEBS Lett. 1991; 287: 211-214Crossref PubMed Scopus (95) Google Scholar). The N-terminal region also contains a “basic-acid-basic” region (9Ebel W. Skinner M.M. Dierksen K.P. Scott J.M. Trempy J.E. J. Bacteriol. 1999; 181: 2236-2243Crossref PubMed Google Scholar). A “sensor and substrate discrimination” domain has been identified between the ATPase and protease domains (10Smith C.K. Baker T.A. Sauer R.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6678-6682Crossref PubMed Scopus (160) Google Scholar). Purified sensor and substrate discrimination domain binds to known Lon substrates (10Smith C.K. Baker T.A. Sauer R.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6678-6682Crossref PubMed Scopus (160) Google Scholar), suggesting that it plays a role in substrate recognition. Although E. coli Lon is thought to be a homo-tetramer or -octamer, recent structural analysis of the proteolytic domain of Lon revealed that it assembles into hexameric rings (11Botos I. Melnikov E.E. Cherry S. Tropea J.E. Khalatova A.G. Rasulova F. Dauter Z. Maurizi M.R. Rotanova T.V. Wlodawer A. Gustchina A. J. Biol. Chem. 2004; 279: 8140-8148Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Lon contributes to the regulation of several important cellular functions, including radiation resistance, cell division, filamentation, capsular polysaccharide production, lysogeny of certain bacteriophages (reviewed in Refs. 12Goldberg A.L. Moerschell R.P. Chung C.H. Maurizi M.R. Methods Enzymol. 1994; 244: 350-375Crossref PubMed Scopus (181) Google Scholar, 13Goldberg A.L. Eur. J. Biochem. 1992; 203: 9-23Crossref PubMed Scopus (415) Google Scholar, 14Gottesman S. Annu. Rev. Genet. 1989; 23: 163-198Crossref PubMed Scopus (120) Google Scholar), and adaptation to nutritional downshift (4Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (297) Google Scholar). E. coli Lon is known to bind nonspecifically to DNA (15Charette M.F. Henderson G.W. Doane L.L. Markovitz A. J. Bacteriol. 1984; 158: 195-201Crossref PubMed Google Scholar). However, human Lon binds specifically to a single-stranded GT-rich DNA sequence of human mitochondrial DNA (16Fu G.K. Markovitz D.M. Biochemistry. 1998; 37: 1905-1909Crossref PubMed Scopus (88) Google Scholar). In addition, Fu et al. (17Fu G.K. Smith M.J. Markovitz D.M. J. Biol. Chem. 1997; 272: 534-538Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) showed that the E. coli Lon binds to a TG-rich site (pets) of the human immunodeficiency virus type2 enhancer. Whether E. coli Lon has a specific binding site on the E. coli chromosome is unclear. It has been suggested that the basic-acid-basic domain is involved in DNA binding (5Chin D.T. Goff S.A. Webster T. Smith T. Goldberg A.L. J. Biol. Chem. 1988; 263: 11718-11728Abstract Full Text PDF PubMed Google Scholar). Another report speculated that the protease domain is involved in DNA binding, because the amino acid sequence of this domain is similar to that of the DNA-binding protein LexA (18Rotanova T.V. Vopr. Med. Khim. 2002; 48: 541-552PubMed Google Scholar). In both cases, the DNA-binding sites of Lon were predicted from its amino acid sequence. Here, our experimental data suggested that both the polyP- and DNA-binding sites of Lon are localized in the ATPase domain. This explains why polyP competes with DNA for binding to Lon. Finally, based on these findings, we discuss the regulatory effects of polyP on the proteolytic and DNA-binding activities of Lon. Protein Purification—Lon protease fused with maltose-binding protein (MBP-Lon) was expressed in E. coli (pMal-Lon) and purified using amylose resin as described by Sonezaki et al. (19Sonezaki S. Kondo A. Oba T. Ishii Y. Kato Y. Nakayama H. Appl. Microbiol. Biotechnol. 1994; 42: 313-318PubMed Google Scholar). MBP-Lon was further purified by Hi Trap Q anion exchange chromatography (Amersham Biosciences). MBP-Lon was eluted from the Hi Trap Q column with a linear gradient of 0–1 m NaCl in buffer A (20 mm Tris-HCl, pH 8.0, 1 mm EDTA, and 20% glycerol). Lon was purified after the cleavage of proteolytic removal of MBP as described previously (19Sonezaki S. Kondo A. Oba T. Ishii Y. Kato Y. Nakayama H. Appl. Microbiol. Biotechnol. 1994; 42: 313-318PubMed Google Scholar). The proteolytically inactive mutant, Lon-S679A, was also expressed as a fusion with MBP (a gift from Dr. S. Sonezaki (Toto Ltd., Kitakyushu, Japan)). MBP-SulA was purified as described previously (20Sonezaki S. Ishii Y. Okita K. Sugino T. Kondo A. Kato Y. Appl. Microbiol. Biotechnol. 1995; 43: 304-309Crossref PubMed Scopus (29) Google Scholar). Ribosomal proteins were purified from isolated ribosomes as follows. Ribosomes were isolated from E. coli MG1655 by ultracentrifugation (21Wickstrom E. Laing L.G. Methods Enzymol. 1988; 164: 238-258Crossref PubMed Scopus (6) Google Scholar). Ribosomal proteins were extracted by incubating the purified ribosomes for 20 h at 4 °C with 4.5 m LiCl and 6 mm β-mercaptoethanol (22Cachia C. Flamion P.J. Schreiber J.P. J. Chromatogr. 1991; 539: 343-353Crossref PubMed Scopus (5) Google Scholar). After precipitating the ribosomal RNAs by centrifugation (5000 × g, 5 min), the supernatant containing ribosomal proteins was dialyzed against buffer B (50 mm ammonium acetate, pH 5.6, 10% glycerol, and 0.15 m NaCl). The supernatant was applied to a carboxymethyl cation exchange column (PerSeptive Biosystems, Framingham, MA), and ribosomal proteins were eluted with a linear gradient of 150–750 mm NaCl in buffer B over 15-column volumes. Some ribosomal proteins were further purified by gel filtration chromatography with Superdex 75 (Amersham Biosciences) in buffer A containing 150 mm NaCl. Degradation of Ribosomal Proteins by Lon—Ribosomal proteins (5 μg) and Lon (1.5 μg) were incubated in a buffer containing 25 mm Tris-HCl, pH 7.8, 4 mm ATP, and 5 mm Mg2+ in the presence and absence of 1.4 μm polyP700 as a polymer at 37 °C and then separated by 12.5% SDS-PAGE. PolyP700 was synthesized by polyP kinase with ATP as a substrate as described previously (4Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (297) Google Scholar). After electrophoresis, ribosomal proteins were stained with SYPRO Orange (Molecular Probes, Eugene, OR) and then visualized with a Typhoon image analyzer (Amersham Biosciences). Construction of Deletion Mutants of Lon—To construct deletion mutants of Lon fused to MBP, a DNA fragment corresponding to Lon 1–713 was amplified using primers F1 and R713 and pMal-Lon (S679A) as the template. The amplified DNA fragment was digested with EcoRI and then inserted into XmnI and EcoRI-digested pMal-c-2x (New England BioLabs, Ontario, Canada). Lon 1–302, Lon 136–784, Lon 272–784, Lon 320–784, Lon 181–302, Lon 272–437, Lon 320–437, Lon 472–616, and Lon 618–775 were constructed with using primers F1 and R302, F136 and R784, F272 and R784, F320 and R784, F181 and R302, F272 and R437, F320 and R437, F472 and R616, and F618 and R775, respectively. DNA sequences of these primers are shown in Table I.Table IDNA sequence of primersPrimerDNA in a PolyP mutants of Lon fused to MBP were expressed in E. coli on of of and 5 of J. T. A Scholar) with mm were by centrifugation and in buffer J. T. A Scholar). After for 5 were separated by 12.5% SDS-PAGE. proteins were to The were with an buffer (50 mm Tris-HCl, pH mm 1 mm and and incubated for h at 37 °C in the same buffer containing PolyP700 with (4Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (297) Google Scholar) was and the was incubated for h at 37 The were with the buffer and then to a The was using the Typhoon image analyzer (Amersham Biosciences). and DNA-binding mutants of Lon fused with MBP were purified using amylose Purified proteolytically inactive MBP-Lon (S679A) or its deletion mutants and were for 5 at in of 25 mm Tris-HCl, pH 7.8, 5 mm and 1 mm The was applied to a and the free was a with a of 25 mm Tris-HCl, pH 7.8, 5 mm and mm NaCl (4Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (297) Google Scholar). The on the corresponding to the amount of was by the DNA-binding a DNA fragment from the was amplified using and as primers and as a and then with J. T. A Scholar). The DNA-binding activity of Lon was as described using DNA of The on the was by of DNA from DNA-Lon inactive Lon fused with MBP was expressed in E. coli After the E. coli were a containing was applied to the amylose The column was with of 20 mm Tris-HCl, pH 8.0, 1 mm EDTA, and 20 and then with of the same buffer containing mm NaCl. After free DNA was completely eluted from the MBP-Lon was eluted with the same buffer containing mm The containing MBP-Lon was extracted with the same of after which DNA was with The DNA was with inserted into the site of and then used to E. from were J. T. A Scholar). DNA and Lon (S679A) μg) were incubated for 5 at in the presence or absence of The were then with and separated by gel J. T. A Scholar). After with DNA was visualized DNA (17Fu G.K. Smith M.J. Markovitz D.M. J. Biol. Chem. 1997; 272: 534-538Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) was synthesized and used as a PolyP the of the presence of polyP, Lon degrades most ribosomal proteins (4Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (297) Google Scholar). We purified several ribosomal proteins of the and and to Lon-mediated degradation in the presence or absence of Lon degraded and in the presence but not in the absence of polyP PolyP also the degradation of purified and but not and not Lon is known to be for the of regulatory proteins, including a cell in response to DNA MBP-SulA was to degradation by Lon in the presence and absence of In to the ribosomal proteins the degradation of the MBP-SulA protein was inhibited in the presence of 1.4 μm polyP our results showed that polyP not Lon-mediated protein degradation and that polyP the substrate of Lon. for the of Ribosomal Protein ribosomal proteins bind to polyP and are degraded by Lon in the presence of polyP (4Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (297) Google Scholar). The MBP-SulA protein not bind to polyP, that polyP binding is for the polyP stimulation of Lon-mediated However, not polyP-binding proteins are degraded by Lon in the presence of polyP and can bind to polyP, but are not degraded by Lon in the presence of it is that polyP binding is but not for the polyP stimulation of Lon-mediated Lon also binds polyP (4Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (297) Google Scholar). polyP first bind to Lon and then the polyP-Lon complex degrades the ribosomal polyP bind to ribosomal proteins and then Lon the protein complex for In the polyP may as a in (reviewed in Refs. A. A. Annu. Rev. Biochem. 1998; PubMed Scopus Google Scholar, W. J. F. E. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Goldberg A.L. Annu. Rev. Biochem. 1996; PubMed Scopus Google Scholar), to ribosomal proteins for degradation by these we examined the of the of on cleavage We that Lon degraded Lon and polyP were together to the of was degradation of was first with polyP the of Lon This that polyP-Lon complex is important for the stimulation of protein degradation and that polyP not as a for Lon-mediated and DNA-binding of Lon the ATPase of sensor and substrate and proteolytic domains the polyP-binding site of Lon, we proteins containing deletion mutants of Lon fused to the terminus of proteins were expressed and to a PolyP was using The results of this experiment showed that the polyP-binding site is localized on Lon between amino and Because Lon also to polyP, the polyP-binding site is to the region between amino and polyP binding appears to be by the ATPase domain. We examined this further using purified Lon deletion The fusion proteins were purified by amylose chromatography and then used in a polyP-binding assay We that the binding activity of the purified Lon was almost same as that of Lon. This the that the polyP-binding site is localized in the ATPase domain of Lon. Although Lon was identified as a DNA-binding protein (5Chin D.T. Goff S.A. Webster T. Smith T. Goldberg A.L. J. Biol. Chem. 1988; 263: 11718-11728Abstract Full Text PDF PubMed Google Scholar, M.F. Henderson G.W. Doane L.L. Markovitz A. J. Bacteriol. 1984; 158: 195-201Crossref PubMed Google Scholar), the of the DNA-binding site has not been We a DNA-binding assay using the purified Lon deletion We used E. coli DNA as a as shown a DNA fragment was with that the DNA-binding site also appears to be localized in the ATPase domain of Lon. Lon to polyP than to the ATPase domain contains both polyP- and DNA-binding polyP may compete with DNA for the binding to Lon. this we the of polyP on the DNA-Lon complex using a assay DNA a complex with Lon, but in the presence of an equimolar amount of polyP, of the DNA-Lon complex was almost completely This suggested that polyP binds to Lon than to DNA. further that the DNA-Lon complex is inhibited in the presence of polyP, we examined the of polyP on MBP-Lon binding to a MBP-Lon was not eluted from the column by a buffer but was eluted the buffer polyP This suggested that polyP can bind to Lon at the same site as DNA of Lon from column with MBP-Lon (50 μg) was applied to a (Amersham Biosciences) column with 20 mm Tris-HCl, pH 8.0, containing mm NaCl. After with of the same MBP-Lon was eluted from the column with the buffer containing μm polyP as a was separated by and MBP-Lon was visualized by the of the using amylose we that purified MBP-Lon contains DNA. This DNA was the MBP-Lon was further purified using an the E. coli were to nutritional which increases polyP level as as 1000-fold (2Rao N.N. Kornberg A. J. Bacteriol. 1996; 178: 1394-1400Crossref PubMed Google Scholar, 3Kuroda A. Murphy H. Cashel M. Kornberg A. J. Biol. Chem. 1997; 272: 21240-21243Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), we a amount of polyP with the amylose MBP-Lon not our results showed that Lon can a complex with DNA in and in Lon forms a complex with polyP it is Lon to E. coli Although Fu et al. (17Fu G.K. Smith M.J. Markovitz D.M. J. Biol. Chem. 1997; 272: 534-538Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) showed that the E. coli Lon binds to a TG-rich site of the human immunodeficiency virus type2 it is whether Lon binds to specific sites on the E. coli this in we and the DNA that were with the amylose Lon. DNA were for DNA sequences to containing and and containing the between and and and and and was the sequences of these DNA Thus, in with et al. (15Charette M.F. Henderson G.W. Doane L.L. Markovitz A. J. Bacteriol. 1984; 158: 195-201Crossref PubMed Google Scholar), it appears that Lon binds nonspecifically to E. coli with from the site of is to to to to to to to to to to to to to to to to to to to to to from the site of is in a We also examined the of and the DNA. We used synthesized TG-rich DNA as a in the DNA-binding The DNA-Lon complex was in the presence of a of the DNA not suggesting that the DNA has affinity Lon than the DNA. PolyP the of Lon by a polyP-Lon is and the of proteins that for degradation are well known in of proteins with to the and is for the degradation of many proteins in A. A. Annu. Rev. Biochem. 1998; PubMed Scopus Google Scholar, W. J. F. E. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Goldberg A.L. Annu. Rev. Biochem. 1996; PubMed Scopus Google Scholar). In the and degradation a unique for the of proteins by Sauer R.T. Biol. PubMed Scopus Google Scholar). we that polyP as a to Lon-mediated protein However, our of suggested that polyP first binds to Lon than to the ribosomal proteins, suggesting that polyP not as a for Lon-mediated We that based on gel the polyP-Lon complex is very J. N. H. and A. a of not Because polyP binds to of Lon (4Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (297) Google Scholar), it be that the polyP-Lon complex is a of the Lon be to the of the polyP-Lon PolyP the DNA-binding of of polyP and DNA binding by Lon mutants that the polyP- and DNA-binding sites of Lon are localized in the ATPase domain. polyP to Lon than which polyP to the DNA in as well as in The ATPase domain is thought to be involved in the important activity (10Smith C.K. Baker T.A. Sauer R.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6678-6682Crossref PubMed Scopus (160) Google Scholar), and DNA has been to the ATPase activity of Lon (15Charette M.F. Henderson G.W. Doane L.L. Markovitz A. J. Bacteriol. 1984; 158: 195-201Crossref PubMed Google Scholar). However, we shown previously that polyP the ATPase activity of Lon in the absence of substrates (4Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (297) Google Scholar). It is a that both of the polyP-Lon and DNA-Lon ATPase activities polyP and DNA the ATPase domain. It be that polyP and DNA binding to the ATPase domain are than of polyP on the and DNA-binding of and Goldberg S.A. Goldberg A.L. J. Biol. Full Text PDF PubMed Google Scholar) that of Lon the of proteins in and is to the We also that E. coli Lon was not Lon is to Sonezaki et al. S. Okita K. Oba T. Ishii Y. Kondo A. Kato Y. Appl. Microbiol. Biotechnol. 1995; PubMed Scopus Google Scholar) speculated that Lon is localized in the complex which its to and proteins also showed that MBP-Lon was from DNA in the presence of but not and that of MBP-Lon at °C its DNA-binding but not its protease suggested that to a of Lon from DNA it to proteins. our data suggested that the of polyP in response to nutritional the polyP-Lon complex is from DNA it can and free ribosomal proteins. It is also that Lon binds to DNA at a site to a regulatory site, to degradation of the regulatory protein. In with this suggested that Lon the level of from the E. coli Markovitz A. J. Bacteriol. PubMed Google Scholar). In this polyP may the of Lon in However, et al. (15Charette M.F. Henderson G.W. Doane L.L. Markovitz A. J. Bacteriol. 1984; 158: 195-201Crossref PubMed Google Scholar), we that E. coli Lon is a DNA-binding protein with low are to the role of Lon in It whether the role of the DNA-binding of Lon is to it from and substrate proteins or to mediate degradation of regulatory proteins. In the in intracellular polyP that occurs during nutritional downshift can the DNA-binding of Lon and its regulation of cellular

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