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A colicin-tolerant Escherichia coli mutant that confers Hfl phenotype carries two mutations in the region coding for the C-terminal domain of FtsH (HflB)
FEMS Microbiology Letters 183 (2000) 115^117
www.fems-microbiology.org
A colicin-tolerant Escherichia coli mutant that confers H£ phenotype carries two mutations in the region coding for the C-terminal domain of FtsH (H£B) Dinah Te¡ a , Simi Koby a , Yoram Shotland a
a;1
, Teru Ogura b , Amos B. Oppenheim
a;
*
Department of Molecular Genetics and Biotechnology, The Hebrew University-Hadassah Medical School, P.O. Box 12272, 91120 Jerusalem, Israel b Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto 862-0976, Japan Received 1 December 1999; accepted 13 December 1999
Abstract An Escherichia coli mutant, ER437, which was originally isolated for colicin tolerance, was found to carry two amino acid changes in the C-terminal portion of FtsH (HflB). These mutations were demonstrated to reduce the ability of FtsH to degrade the phage V CII protein in vivo and in vitro, providing a rationalization for the mutant Hfl phenotype. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Lysis-lysogeny decision ; ATP-dependent protease; AAA protein family
1. Introduction FtsH (H£B) is a membrane-bound Zn2 metalloprotease [1] which is highly conserved; it is found in prokaryotic cells, mitochondria and chloroplasts and is the only known essential ATP-dependent protease in Escherichia coli [2^4]. Among the protein substrates for the E. coli FtsH (H£B) is UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase encoded by the lpxC (envA) gene [3], an enzyme that is involved in lipid A biosynthesis. Mutations a¡ecting FtsH (H£B) levels lead to unbalanced synthesis of major membrane components. Other substrates for FtsH (H£B) include the heat shock sigma factor c32 , the phage V CII, CIII and Xis proteins, SecY, YccA, subunit a of the membrane-embedded F0 part of
* Corresponding author. Tel. : +972 (2) 6757309; Fax: +972 (2) 6757308; E-mail: [email protected] 1 Present address: Washington University School of Medicine, Department of Molecular Microbiology, Campus Box 8230, 660 S. Euclid Ave., St. Louis, MO 63110-1093, USA.
the H -ATPase complex, and SsrA-tagged proteins [1,5^ 11]. Following infection by phage V, the V CII regulatory protein, which activates transcription of the CI repressor from the pRE promoter, is rapidly degraded [12,13]. The V CIII extends the half-life of CII and thereby promotes the lysogenic pathway [14]. The level of CII is ¢nely tuned by the negative phage transcriptional regulators Cro and CI and by rapid proteolysis. Thus, repressor synthesis from pRE is restricted to a very narrow time window (see [15] for detailed analysis). A number of tolZ mutations, selected for colicin tolerance, possess the H£ phenotype. These mutations were found to be located in ftsH (h£B) [16,17]. The molecular mechanism by which a defect in FtsH (H£B) leads to colicin tolerance is not known. The E. coli mutant ER437 was isolated as a temperature-sensitive mutant tolerant to the colicins E2 and E3 [18]. It was later shown to display H£ phenotype preventing the growth of wild-type V and V CIII mutants but allowing plaque formation by V CI and V CII mutants [19]. The fact that expression of CI repressor and integrase following infection of ER437 by phage V is CIII-independent suggested increased CII stability [20]. It was later found that, in this background, the CII protein is stable and is not rapidly degraded [13].
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 6 4 7 - 3
FEMSLE 9219 18-1-00
116
D. Te¡ et al. / FEMS Microbiology Letters 183 (2000) 115^117
bated at 40³C for 30 min, treated with ColE2 at 40³C, diluted and plated. Plates were incubated at 30³C and surviving colonies were counted.
2. Materials and methods 2.1. Sequencing of the ER437 ftsH (h£B) gene A PCR fragment carrying the mutant ftsH gene was generated using oligonucleotides 5P-CGAGATCTATGGCGAAAAACCTAAT-3P, 5P-CGGAATTCTTACTTGTCGCCTAAC-3P and genomic ER437 DNA as template. The PCR product was used to determine the complete sequence of ftsH. 2.2. Cloning of the ftsH (h£B) gene Plasmid pGEX-2T (Pharmacia) was digested with BamHI and EcoRI and ligated to a BglII-EcoRI PCR fragment of the ftsH wild-type or the ER437 mutant genes (using the oligonucleotides described above) to construct GSTFtsH fusion proteins. 2.3. Expression of wild-type and mutant protein fusions The fusion proteins were expressed in strain A8926 carrying an ftsH deletion. IPTG (1 mM) induction was carried out at 20³C for 18 h. 2.4. Puri¢cation of GST-FtsH fusion proteins
3. Results and discussion In order to test if a component of the ER437 mutation is located in the ftsH (h£B) gene, we introduced a plasmid carrying the ftsH (h£B) gene and assayed for phage V growth. It was found that the introduction of a plasmid expressing the ftsH (h£B) gene (pAR145) into the ER437 strain suppressed the H£ phenotype. Both wild-type V and V CIII mutants were able to form plaques on ER437/ pAR145 but not on ER437 (data not shown). These results suggested that a mutation in ftsH (h£B) is responsible for the H£ phenotype of ER437. Sequencing the entire ftsH (h£B) gene of the ER437 mutant, following PCR ampli¢cation, led to the identi¢cation of two amino acid replacements. One of the mutational changes, a CAG to AAG, led to the replacement of a non-conserved glutamine residue by a lysine residue (Q509K). A second mutational change resulted from a double replacement, GAA to ACA, resulting in the replacement of a highly conserved glutamic acid residue by threonine (E551T).
The cells were washed and resuspended in phosphatebu¡ered saline pH 8.0, supplemented with Complete1 EDTA-free protease inhibitors (Boehringer Mannheim # BM1873580) and 20 mM dithiothreitol (DTT). The cells were disrupted by sonication, followed by centrifugation and the soluble GST-FtsH fusion proteins were bound to glutathione agarose beads. The beads were washed, and the fusion proteins were eluted with glutathione. The proteins were stored in a storage bu¡er (200 mM NaCl, 0.5% NP-40, 20 mM monoethanolamine, 1 mM DTT and 30% glycerol) at 370³C. 2.5. Activity of FtsH in vitro The activity of FtsH was determined by following the degradation of CII. The degradation reaction, carried out in two independent experiments, was performed as previously described [6]. A standard reaction (20 Wl) contained 10 pmol of puri¢ed GST-FtsH protein, and 50 pmol CII in 50 mM Tris acetate, 5 mM Mg acetate, 80 mM NaCl, 1.4 mM L-mercaptoethanol, and 5 mM ATP. The reactions were incubated at 42³C and stopped by heating to 95³C for 4 min. 2.6. Transduction experiments The ftsH (h£B) present in ER437 was transduced utilizing the zha-6: :Tn10 marker which is linked to ftsH (h£B). For colicin tolerance tests growing cultures were preincu-
Fig. 1. ER437 FtsH mutant protein is defective in the proteolysis of CII. A: Puri¢ed wild-type or ER437 FtsH proteins (10 pmol) were incubated with CII (50 pmol, 100%) under standard conditions [6]. The proteins were separated on 15% SDS-PAGE gels and the CII protein was identi¢ed by silver staining. B: Degradation as a function of time was determined by computer imaging (NIH Image program). The experiments were repeated twice and representative results are shown. Filled squares, wild-type ; open squares, ER437.
FEMSLE 9219 18-1-00
D. Te¡ et al. / FEMS Microbiology Letters 183 (2000) 115^117
In order to demonstrate directly that the mutations in ftsH (h£B) are responsible for colicin tolerance we transduced the ftsH (h£B) present in ER437 utilizing the zha6: :Tn10 marker which is linked to ftsH (h£B). Both temperature-sensitive growth and colicin tolerance characteristics were 18% cotransducible with zha-6: :Tn10. These results demonstrate that the mutation in ftsH (h£B) is responsible for both colicin tolerance and phenotypes. To test directly the activity of this mutant FtsH (H£B) protein, the ftsH (h£B) gene of the ER437 was cloned and expressed as a GST-FtsHER437 protein fusion. We puri¢ed both wild-type and ER437 GST-FtsH proteins and tested their ability to degrade CII in vitro. Wild-type GST-FtsH degrades CII with a half-life of 2^3 min at 42³C (Fig. 1). In contrast, the GST-FtsHER437 was unable to degrade the CII protein. These results demonstrate that the FtsH (H£B) protein present in ER437 is inactive. The functional defect induced by the ER437 mutation is not known. However, a number of mutations were found to be located at the C-terminal domain of FtsH (H£B). The ER437 mutation E551T is located close to the H539R change introduced by the h£B29 mutation which reduces the rate of CII proteolysis in vivo by about 2^3-fold [21]. In addition, a set of mutations L567R, L574R, L574A and L581R were also found to interfere with FtsH activity (our unpublished results). The latter mutations are all located in a small region that we have recently demonstrated to form a coiled-coil structure. Our early studies on the control of phage V gene expression in the ER437 strain demonstrated that CII-dependent repressor synthesis, from the pRE promoter, and the expression of exonuclease, from the pL promoter, were not turned o¡ [19]. It was suggested that the ER437 interfered with the synthesis or function of Cro. It is highly likely that the elevated transcription from the pRE promoter (resulting from the increased stability of CII) prevents Cro synthesis. This inhibition could be accomplished by the CI transcript, originated from the pRE promoter, acting as antisense RNA for the cro mRNA.
[4] [5]
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15] [16]
[17]
References [18] [1] Tomoyasu, T., Gamer, J., Bukau, B., Kanemori, M., Mori, H., Rutman, A.J., Oppenheim, A.B., Yura, T., Yamanaka, K., Niki, H., Higara, S. and Ogura, T. (1995) Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heatshock transcription factor sigma 32. EMBO J. 14, 2551^2560. [2] Tomoyasu, T., Yuki, T., Morimura, S., Mori, H., Yamanaka, K., Niki, H., Hiraga, S. and Ogura, T. (1993) The Escherichia coli FtsH protein is a prokaryotic member of a protein family of putative ATPases involved in membrane functions, cell cycle control, and gene expression. J. Bacteriol. 175, 1344^1351. [3] Ogura, T., Inoue, K., Tatsuta, T., Suzaki, T., Karata, K., Young, K., Su, L.H., Fierke, C.A., Jackman, J.E., Raetz, C.R., Coleman, J.,
[19]
[20]
[21]
117
Tomoyasu, T. and Matsuzawa, H. (1999) Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (H£B) in Escherichia coli. Mol. Microbiol. 31, 833^844. Schumann, W. (1999) FtsH a single-chain charonin? FEMS Microbiol. Rev. 23, 1^11. Herman, C., Thevenet, D., D'Ari, R. and Bouloc, P. (1995) Degradation of sigma 32, the heat shock regulator in Escherichia coli, is governed by H£B. Proc. Natl. Acad. Sci. USA 92, 3516^3520. Shotland, Y., Koby, S., Te¡, D., Mansur, N., Oren, D.A., Tatematsu, K., Tomoyasu, T., Kessel, M., Bukau, B., Ogura, T. and Oppenheim, A.B. (1997) Proteolysis of the phage lambda CII regulatory protein by FtsH (H£B) of Escherichia coli. Mol. Microbiol. 24, 1303^1310. Herman, C., Thevenet, D., D'Ari, R. and Bouloc, P. (1997) The H£B protease of Escherichia coli degrades its inhibitor lambda CIII. J. Bacteriol. 179, 358^363. Kihara, A., Akiyama, Y. and Ito, K. (1998) Di¡erent pathways for protein degradation by the FtsH/H£KC membrane-embedded protease complex: an implication from the interference by a mutant form of a new substrate protein, YccA. J. Mol. Biol. 279, 175^ 188. Herman, C., Thevenet, D., Bouloc, P., Walker, G.C. and D'Ari, R. (1998) Degradation of carboxy-terminal-tagged cytoplasmic proteins by the Escherichia coli protease H£B (FtsH). Genes Dev. 12, 1348^ 1355. Akiyama, Y., Kihara, A. and Ito, K. (1996) Subunit a of proton ATPase F0 sector is a substrate of the FtsH protease in Escherichia coli. FEBS Lett. 399, 26^28. Le¡ers, G.G. and Gottesman, S. (1998) Lambda Xis degradation in vivo by Lon and FtsH. J. Bacteriol. 180, 1573^1577. Hoyt, M.A., Knight, D.M., Das, A., Miller, H.I. and Echols, H. (1982) Control of phage V development by stability and synthesis of CII protein : role of the viral cIII and host h£A, himA, and himD genes. Cell 31, 565^573. Rattray, A., Altuvia, S., Mahajna, J., Oppenheim, A.B. and Gottesman, M. (1984) Control of bacteriophage lambda CII activity by bacteriophage and host functions. J. Bacteriol. 159, 238^242. Ptashne, M. (1992) in: A Genetic Switch: Phage V and Higher Organisms. Cell Press and Blackwell Scienti¢c Publications, Cambridge, MA. McAdams, H.H. and Shapiro, L. (1995) Circuit simulation of genetic networks. Science 269, 650^656. Qu, J.N., Makino, S.I., Adachi, H., Koyama, Y., Akiyama, Y., Ito, K., Tomoyasu, T., Ogura, T. and Matsuzawa, H. (1996) The tolZ gene of Escherichia coli is identi¢ed as the ftsH gene. J. Bacteriol. 178, 3457^3461. Makino, S., Qu, J.N., Uemori, K., Ichikawa, H., Ogura, T. and Matsuzawa, H. (1997) A silent mutation in the ftsH gene of Escherichia coli that a¡ects FtsH protein production and colicin tolerance. Mol. Gen. Genet. 254, 578^583. Nomura, M. and Witten, C. (1967) Interaction of colicins with bacterial cells. III. Colicin tolerant mutations in Escherichia coli. J. Bacteriol. 94, 1093^1111. Oppenheim, A., Honigman, A. and Oppenheim, A.B. (1974) Interference with phage lambda cro gene function by a colicin-tolerant Escherichia coli mutant. Virology 61, 1^10. Belfort, M., Kass, N., Oppenheim, A., Katzir, N. and Oppenheim, A.B. (1977) Repressor and Int synthesis of bacteriophage V in the E. coli host mutant ER437. Mol. Gen. Genet. 155, 347^349. Banuett, F., Hoyt, M.A., McFarlane, L., Echols, H. and Herskowitz, I. (1986) h£B, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda CII protein. J. Mol. Biol. 187, 213^ 224.
FEMSLE 9219 18-1-00
www.fems-microbiology.org
A colicin-tolerant Escherichia coli mutant that confers H£ phenotype carries two mutations in the region coding for the C-terminal domain of FtsH (H£B) Dinah Te¡ a , Simi Koby a , Yoram Shotland a
a;1
, Teru Ogura b , Amos B. Oppenheim
a;
*
Department of Molecular Genetics and Biotechnology, The Hebrew University-Hadassah Medical School, P.O. Box 12272, 91120 Jerusalem, Israel b Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto 862-0976, Japan Received 1 December 1999; accepted 13 December 1999
Abstract An Escherichia coli mutant, ER437, which was originally isolated for colicin tolerance, was found to carry two amino acid changes in the C-terminal portion of FtsH (HflB). These mutations were demonstrated to reduce the ability of FtsH to degrade the phage V CII protein in vivo and in vitro, providing a rationalization for the mutant Hfl phenotype. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Lysis-lysogeny decision ; ATP-dependent protease; AAA protein family
1. Introduction FtsH (H£B) is a membrane-bound Zn2 metalloprotease [1] which is highly conserved; it is found in prokaryotic cells, mitochondria and chloroplasts and is the only known essential ATP-dependent protease in Escherichia coli [2^4]. Among the protein substrates for the E. coli FtsH (H£B) is UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase encoded by the lpxC (envA) gene [3], an enzyme that is involved in lipid A biosynthesis. Mutations a¡ecting FtsH (H£B) levels lead to unbalanced synthesis of major membrane components. Other substrates for FtsH (H£B) include the heat shock sigma factor c32 , the phage V CII, CIII and Xis proteins, SecY, YccA, subunit a of the membrane-embedded F0 part of
* Corresponding author. Tel. : +972 (2) 6757309; Fax: +972 (2) 6757308; E-mail: [email protected] 1 Present address: Washington University School of Medicine, Department of Molecular Microbiology, Campus Box 8230, 660 S. Euclid Ave., St. Louis, MO 63110-1093, USA.
the H -ATPase complex, and SsrA-tagged proteins [1,5^ 11]. Following infection by phage V, the V CII regulatory protein, which activates transcription of the CI repressor from the pRE promoter, is rapidly degraded [12,13]. The V CIII extends the half-life of CII and thereby promotes the lysogenic pathway [14]. The level of CII is ¢nely tuned by the negative phage transcriptional regulators Cro and CI and by rapid proteolysis. Thus, repressor synthesis from pRE is restricted to a very narrow time window (see [15] for detailed analysis). A number of tolZ mutations, selected for colicin tolerance, possess the H£ phenotype. These mutations were found to be located in ftsH (h£B) [16,17]. The molecular mechanism by which a defect in FtsH (H£B) leads to colicin tolerance is not known. The E. coli mutant ER437 was isolated as a temperature-sensitive mutant tolerant to the colicins E2 and E3 [18]. It was later shown to display H£ phenotype preventing the growth of wild-type V and V CIII mutants but allowing plaque formation by V CI and V CII mutants [19]. The fact that expression of CI repressor and integrase following infection of ER437 by phage V is CIII-independent suggested increased CII stability [20]. It was later found that, in this background, the CII protein is stable and is not rapidly degraded [13].
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 6 4 7 - 3
FEMSLE 9219 18-1-00
116
D. Te¡ et al. / FEMS Microbiology Letters 183 (2000) 115^117
bated at 40³C for 30 min, treated with ColE2 at 40³C, diluted and plated. Plates were incubated at 30³C and surviving colonies were counted.
2. Materials and methods 2.1. Sequencing of the ER437 ftsH (h£B) gene A PCR fragment carrying the mutant ftsH gene was generated using oligonucleotides 5P-CGAGATCTATGGCGAAAAACCTAAT-3P, 5P-CGGAATTCTTACTTGTCGCCTAAC-3P and genomic ER437 DNA as template. The PCR product was used to determine the complete sequence of ftsH. 2.2. Cloning of the ftsH (h£B) gene Plasmid pGEX-2T (Pharmacia) was digested with BamHI and EcoRI and ligated to a BglII-EcoRI PCR fragment of the ftsH wild-type or the ER437 mutant genes (using the oligonucleotides described above) to construct GSTFtsH fusion proteins. 2.3. Expression of wild-type and mutant protein fusions The fusion proteins were expressed in strain A8926 carrying an ftsH deletion. IPTG (1 mM) induction was carried out at 20³C for 18 h. 2.4. Puri¢cation of GST-FtsH fusion proteins
3. Results and discussion In order to test if a component of the ER437 mutation is located in the ftsH (h£B) gene, we introduced a plasmid carrying the ftsH (h£B) gene and assayed for phage V growth. It was found that the introduction of a plasmid expressing the ftsH (h£B) gene (pAR145) into the ER437 strain suppressed the H£ phenotype. Both wild-type V and V CIII mutants were able to form plaques on ER437/ pAR145 but not on ER437 (data not shown). These results suggested that a mutation in ftsH (h£B) is responsible for the H£ phenotype of ER437. Sequencing the entire ftsH (h£B) gene of the ER437 mutant, following PCR ampli¢cation, led to the identi¢cation of two amino acid replacements. One of the mutational changes, a CAG to AAG, led to the replacement of a non-conserved glutamine residue by a lysine residue (Q509K). A second mutational change resulted from a double replacement, GAA to ACA, resulting in the replacement of a highly conserved glutamic acid residue by threonine (E551T).
The cells were washed and resuspended in phosphatebu¡ered saline pH 8.0, supplemented with Complete1 EDTA-free protease inhibitors (Boehringer Mannheim # BM1873580) and 20 mM dithiothreitol (DTT). The cells were disrupted by sonication, followed by centrifugation and the soluble GST-FtsH fusion proteins were bound to glutathione agarose beads. The beads were washed, and the fusion proteins were eluted with glutathione. The proteins were stored in a storage bu¡er (200 mM NaCl, 0.5% NP-40, 20 mM monoethanolamine, 1 mM DTT and 30% glycerol) at 370³C. 2.5. Activity of FtsH in vitro The activity of FtsH was determined by following the degradation of CII. The degradation reaction, carried out in two independent experiments, was performed as previously described [6]. A standard reaction (20 Wl) contained 10 pmol of puri¢ed GST-FtsH protein, and 50 pmol CII in 50 mM Tris acetate, 5 mM Mg acetate, 80 mM NaCl, 1.4 mM L-mercaptoethanol, and 5 mM ATP. The reactions were incubated at 42³C and stopped by heating to 95³C for 4 min. 2.6. Transduction experiments The ftsH (h£B) present in ER437 was transduced utilizing the zha-6: :Tn10 marker which is linked to ftsH (h£B). For colicin tolerance tests growing cultures were preincu-
Fig. 1. ER437 FtsH mutant protein is defective in the proteolysis of CII. A: Puri¢ed wild-type or ER437 FtsH proteins (10 pmol) were incubated with CII (50 pmol, 100%) under standard conditions [6]. The proteins were separated on 15% SDS-PAGE gels and the CII protein was identi¢ed by silver staining. B: Degradation as a function of time was determined by computer imaging (NIH Image program). The experiments were repeated twice and representative results are shown. Filled squares, wild-type ; open squares, ER437.
FEMSLE 9219 18-1-00
D. Te¡ et al. / FEMS Microbiology Letters 183 (2000) 115^117
In order to demonstrate directly that the mutations in ftsH (h£B) are responsible for colicin tolerance we transduced the ftsH (h£B) present in ER437 utilizing the zha6: :Tn10 marker which is linked to ftsH (h£B). Both temperature-sensitive growth and colicin tolerance characteristics were 18% cotransducible with zha-6: :Tn10. These results demonstrate that the mutation in ftsH (h£B) is responsible for both colicin tolerance and phenotypes. To test directly the activity of this mutant FtsH (H£B) protein, the ftsH (h£B) gene of the ER437 was cloned and expressed as a GST-FtsHER437 protein fusion. We puri¢ed both wild-type and ER437 GST-FtsH proteins and tested their ability to degrade CII in vitro. Wild-type GST-FtsH degrades CII with a half-life of 2^3 min at 42³C (Fig. 1). In contrast, the GST-FtsHER437 was unable to degrade the CII protein. These results demonstrate that the FtsH (H£B) protein present in ER437 is inactive. The functional defect induced by the ER437 mutation is not known. However, a number of mutations were found to be located at the C-terminal domain of FtsH (H£B). The ER437 mutation E551T is located close to the H539R change introduced by the h£B29 mutation which reduces the rate of CII proteolysis in vivo by about 2^3-fold [21]. In addition, a set of mutations L567R, L574R, L574A and L581R were also found to interfere with FtsH activity (our unpublished results). The latter mutations are all located in a small region that we have recently demonstrated to form a coiled-coil structure. Our early studies on the control of phage V gene expression in the ER437 strain demonstrated that CII-dependent repressor synthesis, from the pRE promoter, and the expression of exonuclease, from the pL promoter, were not turned o¡ [19]. It was suggested that the ER437 interfered with the synthesis or function of Cro. It is highly likely that the elevated transcription from the pRE promoter (resulting from the increased stability of CII) prevents Cro synthesis. This inhibition could be accomplished by the CI transcript, originated from the pRE promoter, acting as antisense RNA for the cro mRNA.
[4] [5]
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15] [16]
[17]
References [18] [1] Tomoyasu, T., Gamer, J., Bukau, B., Kanemori, M., Mori, H., Rutman, A.J., Oppenheim, A.B., Yura, T., Yamanaka, K., Niki, H., Higara, S. and Ogura, T. (1995) Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heatshock transcription factor sigma 32. EMBO J. 14, 2551^2560. [2] Tomoyasu, T., Yuki, T., Morimura, S., Mori, H., Yamanaka, K., Niki, H., Hiraga, S. and Ogura, T. (1993) The Escherichia coli FtsH protein is a prokaryotic member of a protein family of putative ATPases involved in membrane functions, cell cycle control, and gene expression. J. Bacteriol. 175, 1344^1351. [3] Ogura, T., Inoue, K., Tatsuta, T., Suzaki, T., Karata, K., Young, K., Su, L.H., Fierke, C.A., Jackman, J.E., Raetz, C.R., Coleman, J.,
[19]
[20]
[21]
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