[26] A T5 promoter-based transcription-translation system for the analysis of proteins in Vitro and in Vivo

Bujard, Hermann; Gentz, Reiner; Lanzer, Michael; Stueber, Dietrich; Mueller, Michael; Ibrahimi, Ibrahim; Haeuptle, Marie-Therese; Dobberstein, Bernhard

doi:10.1016/0076-6879(87)55028-5

Cited by 258 publications

(166 citation statements)

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“…(a) pUHE-24S, a modified version of pUHE-24 from which one NcoI site was removed, leaving a unique NcoI site in the translation start codon. pUHE-24 is a descendant of pDS (Deuschle et al, 1986) containing a T5 RBS (Bujard et al, 1987), two tandem copies of lacO and the early T7 (s 70 -activated) promoter P A1 (utilizing the usual E. coli RNA polymerase). (b) pRM4-C and pRM4-RC, which carry cyt1Aa and p20+cyt1Aa, respectively (Manasherob et al, 2001).…”

Section: Methodsmentioning

confidence: 99%

Compaction of the Escherichia coli nucleoid caused by Cyt1Aa

et al. 2003

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Compaction of the Escherichia coli nucleoid in the cell's centre was associated with the loss of colony-forming ability; these effects were caused by induction of Cyt1Aa, the cytotoxic 27 kDa protein from Bacillus thuringiensis subsp. israelensis. Cyt1Aa-affected compaction of the nucleoids was delayed but eventually more intense than compaction caused by chloramphenicol. The possibility that small, compact nucleoids in Cyt1Aa-expressing cells resulted in DNA replication run-out and segregation following cell division was ruled out by measuring relative nucleoid length. Treatments with membrane-perforating substances other than Cyt1Aa did not cause such compaction of the nucleoids, but rather the nucleoids overexpanded to occupy nearly all of the cell volume. These findings support the suggestion that, in addition to its perforating ability, Cyt1Aa causes specific disruption of nucleoid associations with the cytoplasmic membrane. In situ immunofluorescence labelling with Alexa did not demonstrate a great amount of Cyt1Aa associated with the membrane. Clear separation between Alexa-labelled Cyt1Aa and 49,6-diamidino-2-phenylindole (DAPI)-stained DNA indicates that the nucleoid does not bind Cyt1Aa. Around 2 h after induction, nucleoids in Cyt1Aa-expressing cells started to decompact and expanded to fill the whole cell volume, most likely due to partial cell lysis without massive peptidoglycan destruction. INTRODUCTIONDuring sporulation, various subspecies of the Grampositive soil bacterium Bacillus thuringiensis produce large amounts of insecticidal crystal proteins (ICP), the so-called d-endotoxins , each toxic against larvae of a different group of insects. The ICP of B. thuringiensis subsp. israelensis is specific against the larvae of mosquitoes and black flies (Goldberg & Margalit, 1977;Margalith & Ben-Dov, 2000), vectors of many human infectious diseases (Service, 1986). The crystal is composed of four major polypeptides, Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa (of 125, 135, 68 and 28 kDa, respectively), which are encoded by genes carried on the~130 kDa plasmid nicknamed pBtoxis (Ben-Dov et al., 1999). Cyt1Aa, which is not homologous to any of the known Cry toxins , is the most prominent of the four polypeptides, but it is less specific than Cry4Aa, Cry4Ba and Cry11Aa, haemolytic and cytotoxic in vitro (Thomas & Ellar, 1983b;Drobniewski & Ellar, 1988;Hofte & Whiteley, 1989). The broad cytolytic activity of Cyt1Aa has been attributed to its hydrophobicity and ability to bind zwitterionic phospholipids (Thomas & Ellar, 1983a, b). Cyt1Aa is inserted into the membrane to create cation-selective single channels of 1-2 nm in diameter, leading to colloid-osmotic lysis (Knowles & Ellar, 1987;Knowles et al., 1989), and induces leakage of low-molecular-mass substances from lipid vesicles (Drobniewski & Ellar, 1988.Seven cytolytic, mosquitocidal-specific toxins are currently known (Earp & Ellar, 1987;Drobniewski & Ellar, 1989;Yu et al., 1991;Koni & Ellar, 1993;Cheong & Gill, 1997;Guerchicoff et al., 1997;Thiery et al., 199...

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Section: Methodsmentioning

confidence: 99%

Compaction of the Escherichia coli nucleoid caused by Cyt1Aa

et al. 2003

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“…M13mp18 was used as a vector for site-directed mutagenesis. The plasmid pQE30 (Qiagen Inc.) (11), containing the highly efficient T5 promoter and His 6 tag coding sequence, was used as the expression vector to overexpress the wild-type and the mutated GCT-coding genes for kinetic analysis. Plasmid pET11a, from Novagen, was used for expression for NMR analysis.…”

Section: Methodsmentioning

confidence: 99%

Identification of Functional Conserved Residues of CTP:glycerol-3-phosphate Cytidylyltransferase

Park¹,

Gee²,

Sanker³

et al. 1997

Journal of Biological Chemistry

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The CTP:glycerol-3-phosphate cytidylyltransferase (GCT) of Bacillus subtilis has been shown to be similar in primary structure to the CTP:phosphocholine cytidylyltransferases of several organisms. To identify the residues of this cytidylyltransferase family that function in catalysis, the conserved hydrophilic amino acid residues plus a conserved tryptophan of the GCT were mutated to alanine. The most dramatic losses in activity occurred with H14A and H17A; these histidine residues are part of an HXGH sequence similar to that found in class I aminoacyl-tRNA synthetases. The k cat values for H14A and H17A were decreased by factors of 5 ؋ 10 ؊5 and 4 ؋ 10 ؊4 , respectively, with no significant change in K m values. Asp-11, which is found near the HXGH sequence in the cytidylyltransferases but not aminoacyltRNA synthetases, was also important for activity, with the D11A mutation decreasing activity by a factor of 2 ؋ 10 ؊3 . Several residues found in the sequence RTEGISTT, a signature sequence for this cytidylyltransferase family, as well as other isolated residues were also shown to be important for activity, with k cat values decreasing by factors of 0.14 -4 ؋ 10 ؊4 . The K m values of three mutant enzymes, D38A, W74A, and D94A, for both CTP and glycerol-3-phosphate were 6 -130-fold higher than that of the wild-type enzyme. Mutant enzymes were analyzed by two-dimensional NMR to determine if the overall structures of the enzymes were intact. One of the mutant enzymes, D66A, was defective in overall structure, but several of the others, including H14A and H17A, were not. These results indicate that His-14 and His-17 play a role in catalysis and suggest that their role is similar to the role of the His residues in the HXGH sequence in class I aminoacyl-tRNA synthetases, i.e. to stabilize a pentacoordinate transition state.The CTP:glycerol-3-phosphate cytidylyltransferase (GCT) 1 from Bacillus subtilis catalyzes the formation of CDP-glycerol and pyrophosphate from CTP and glycerol-3-phosphate. CDPglycerol then serves as a principal precursor required for biosynthesis of poly(glycerol phosphate), the major teichoic acid found in the bacterial cell wall (1, 2). GCT appears to be a member of a cytidylyltransferase family that includes CTP: phosphocholine cytidylyltransferases (CCT), a key regulatory enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis in higher eukaryotes (3-5). Fig. 1 shows a comparison of the deduced amino acid sequences of several cytidylyltransferases: presumed GCTs from Staphylococcus aureus and Streptomyces wedmorensis, GCT from B. subtilis, CCTs from a variety of organisms, and ethanolamine phosphate cytidylyltransferase (ECT) from yeast. This comparison reveals a number of residues that are conserved among these three enzymes (6). These conserved amino acids are contained within the catalytic core of the CCTs (7). Conservation of these amino acids suggests that they may be required for functions such as catalysis, recognition of substrates, structural integrity, or association ...

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“…This alteration was obtained by point-specifically mutating the pLHCP expression clone XLHCP-2 (Paulsen et al, 1990). This clone contains the coding sequence of the AB80 gene from pea (Cashmore, 1984), called lhb1*2 according to the nomenclature suggested by Jansson et al (1992), inserted into the bacterial expression vector pDS12-RBSII (Bujard et al, 1987). Mutagenesis was performed as described earlier (Paulsen and Kuttkat, 1993).…”

Section: Materials and Methods Site-specific Mutagenesis Of Plhcpmentioning

confidence: 99%

Pigments induce folding of light‐harvesting chlorophyll a/b‐binding protein

Paulsen

Finkenzeller

Kühlein

1993

European Journal of Biochemistry

186

131

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The conformational behaviour of the light-harvesting chlorophyll &-binding protein (LHCP), the apoprotein of the major light-harvesting complex (LHCII) of photosystem I1 in plants, has been studied. According to the circular dichroism in the ultraviolet range measured with isolated LHCII, the protein in the complex adopts a folded structure with a high content of a helix (about 60%), whereas the non-pigmented, solubilized protein has a less ordered structure (about 20% a helix).LHCP-pigment complexes that have been reconstituted from the overexpressed protein and isolated pigments in the presence of detergents display a protein CD signal similar to that of authentic LHCII, indicating that LHCP folds into the native structure during the reconstitution procedure. Renaturation of LHCP in these experiments is dependent on the presence of pigments and the formation of stable LHCP-pigment complexes. Pigment-induced engagement of LHCP in a compact structure has also been shown by two additional experimental approaches. (a) Upon complex formation, LHCP or its precursor (pLHCP) becomes resistant to trypsin digestion with the exception of an N-terminal segment of the protein; the same protection of LHCP is known to occur in intact thylakoids. (b) Pigment binding renders a cysteine residue within the N-proximal hydrophobic domain of the protein as well as a newly introduced cysteine four amino acid positions from the C terminus inaccessible to modification with a sulfhydryl-specific label whereas the N terminus stays susceptible to specific labelling. These observations support the notion that only the N terminus protrudes from a compact protein-pigment structure in LHCII. The fact that the major part of LHCP is trypsin-resistant in pigmented complexes reconstituted in the absence of a membrane or even lipids justifies caution in using protection against trypsin as a criterion for the integration of LHCP into the thylakoid membrane.The efficiency of photosynthesis is greatly enhanced by antenna or light-harvesting complexes which collect light energy and transduce it to the photosynthetic reaction centers. The most prominent antenna in higher plants is the major light-harvesting complex of photosystem I1 (LHCII) comprising about half of the total chlorophyll in the thylakoid membrane. LHCII consists of an apoprotein (LHCP) of 25 -28 kDa and pigments, i.e. 14 or 15 molecules chlorophyll a and b and 2 -4 molecules xanthophyll bound/protein monomer (Bassi et al., 1990; Peter and Thornber, 1991). These pigments are non-covalently bound to the apoprotein. Little is known about the molecular interactions between protein and pigments. The light-harvesting function of LHCII requires that captured light quanta must be rapidly transferred between pigments within an antenna to be efficiently delivered at a reaction center (Sauer, 1986). Very rapid energy transfer between antenna chlorophylls has been measured by Abbreviations. LHCII, major light-harvesting complex of photosystem 11; LHCP, light-harvesting chlorophyll &-binding protei...

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[26] A T5 promoter-based transcription-translation system for the analysis of proteins in Vitro and in Vivo

Cited by 258 publications

References 18 publications

Compaction of the Escherichia coli nucleoid caused by Cyt1Aa

Compaction of the Escherichia coli nucleoid caused by Cyt1Aa

Identification of Functional Conserved Residues of CTP:glycerol-3-phosphate Cytidylyltransferase

Pigments induce folding of light‐harvesting chlorophyll a/b‐binding protein

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