Studies on the structure and function of subtilisin E by protein engineering.
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Amino Acid Sequence↗
Biomedical subjects
M Inouye
Publications and source records attributed to M Inouye.
Neither lipid modification nor processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in Escherichia coli.
The relationship between the modification and processing of prolipoprotein and the formation of murein-bound lipoprotein has been investigated using Escherichia coli mutants altered in the signal sequence of prolipoprotein and an E. coli strain producing OmpF-Lpp hybrid protein. The glyceride-modified prolipoprotein in mutant lppT20 and in globomycin-treated wild-type strain were covalently attached to the peptidoglycan. Likewise, the unmodified prolipoproteins in mutants lppL20, lppV20, and lppG21 were attached to the peptidoglycan. The OmpF-Lpp hybrid protein that is processed but not modified with lipid due to the absence of the cysteine-containing modification site in the hybrid protein was also covalently linked to the peptidoglycan. These results indicate that neither lipid modification nor the processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in E. coli. In contrast, introduction of a charged amino acid residue such as Asp or Arg at the 14th position of prolipoprotein affected not only the lipid modification and processing of the mutant prolipoprotein but also the formation of murein-bound lipoprotein. Replacement of the Gly14 with Glu or Lys partially affected the lipid modification and processing of prolipoprotein; the peptidoglycan of the lppE14 and lppK14 mutants contained a reduced amount of mature lipoprotein but no mutant prolipoprotein. In addition, lpp mutants A20I23I24 and A20I23K24 were found to be defective in both lipid modification/processing of prolipoprotein and the formation of murein-bound lipoprotein. The defective formation of murein-bound lipoprotein in the latter mutants may be related to an alteration in the secondary structure at the modification/processing site of the mutant prolipoproteins.
Amino Acid Sequence↗
Functional analysis of the intramolecular chaperone. Mutational hot spots in the subtilisin pro-peptide and a second-site suppressor mutation within the subtilisin molecule.
The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis. The synthetic pro-peptide has been shown to be capable of guiding the proper folding of denatured subtilisin to enzymatically active enzyme. Thus the pro-peptide serves as an intramolecular chaperone, which is removed by an autoprocessing reaction after the completion of the folding. With use of localized polymerase chain reaction random mutagenesis a total of 25 amino acid substitution mutations that affected subtilisin activities were isolated. These mutations occurred in a high frequency at the hydrophobic regions of the pro-peptide. For one of the mutations, M(-60)T, a second-site suppressor mutation, S(188)L, was isolated within the mature region. These results suggest that the pro-peptide consists of a few functional regions which interact with specific regions of the mature region of subtilisin during the folding process.
Amino Acid Sequence↗
Cold-sensitive growth and decreased GTP-hydrolytic activity from substitution of Pro17 for Val in Era, an essential Escherichia coli GTPase.
A substitution mutation of Pro17 by Val (P17V) was constructed in the guanine nucleotide binding domain of Era, an essential protein in Escherichia coli. The mutation is analogous to the oncogenic activating allele at position 12 in the GTP-binding domain of p21ras. The phenotype of this mutant was analysed in a strain which exclusively expressed the mutant protein (Era-V17) in null allele chromosomal background (era1: :kan). The strain was found to be cold-sensitive for growth. Mutant Era-V17 purified from the strain was cold-sensitive for GTP-hydrolytic activity, suggesting that the GTPase activity of Era is required for cell growth since the P17V mutation resulted in both cold-sensitive growth of cells and cold-labile GTPase activity of the purified protein.
Bacterial Proteins↗
Yeast NSR1 protein that has structural similarity to mammalian nucleolin is involved in pre-rRNA processing.
We have identified a yeast gene encoding a protein structurally similar to mammalian nucleolin. The gene was previously cloned as a cold shock-inducible gene and found to be identical to yeast NSR1 gene, which encodes a protein that has been reported to bind sequences required for nuclear localization of protein. The carboxyl-terminal half of NSR1, consisting of two tandemly repeated putative RNA-binding domains and a glycine/arginine-rich domain, has 37% amino acid sequence identity with the same part of mammalian nucleolin, while no sequence similarities are found between their amino-terminal regions. Although a null mutation of the NSR1 gene was not lethal, it caused a severe defect on growth. Pulse-labeling analysis revealed that the nsr1 strain had reduced levels of 18 S rRNA and accumulated 35 S pre-rRNA compared with the wild-type strain. The level of 25 S rRNA was also slightly reduced in the nsr1 strain. Pulse-chase labeling experiments showed slow processing of 35 S pre-rRNA and impaired methylation of 18 S rRNA. The ratio of 40 S to 60 S ribosomal subunits in the nsr1 strain is significantly reduced and is consistent with impaired synthesis of 18 S rRNA. The results indicate that NSR1 is involved in pre-rRNA processing and ribosome biosynthesis in yeast.
Amino Acid Sequence↗
Cold shock induction of yeast NSR1 protein and its role in pre-rRNA processing.
Yeast NSR1 protein is structurally related to mammalian nucleolin and is involved in ribosome synthesis as described in the preceding paper (Kondo, K., and Inouye, M. (1992) J. Biol. Chem. 267, 16252-16258). We report here the regulation of NSR1 gene expression and the effect of nsr1 deletion on growth and pre-rRNA processing after cold shock. A basal level of the transcript was detected at 30 degrees C only in exponentially growing cells and increased approximately 3-fold after cold shock to 10 degrees C. NSR1 protein level also increased about 3-fold after the shock. The nsr1 deletion caused a severe growth defect at low temperatures as well as a long growth lag after temperature shift from 30 to 10 degrees C. Northern hybridization analysis demonstrated that cold shock led to a rapid decrease in the amounts of 27 S, 20 S, and 7 S rRNA precursors in the nsr1 strain, although at 30 degrees C a major defect was observed only in 20 S pre-rRNA synthesis. Pulse-chase labeling of rRNA showed that pre-rRNA processing in the nsr1 strain was greatly impaired after cold shock. These results demonstrate that the NSR1 protein is required for normal pre-rRNA processing and cell growth in yeast when cells are exposed to abrupt temperature drop.
Base Sequence↗
Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli.
msDNA-Ec67 is produced in a clinical strain of Escherichia coli and composed of a 67-base single-stranded DNA, which is linked to the 2'-OH group of the 15th rG residue of a 58-base RNA molecule by a 2',5'-phosphodiester linkage (Lampson, B. C., Sun, J., Hsu, M.-Y., Vallejo-Ramirez, J., Inouye, S., and Inouye, M. (1989) Science 243, 1033-1038). The production of msDNA-Ec67 is dependent upon retron-Ec67, which consists of the msr-msd region and the gene for reverse transcriptase (RT). These two elements were separately cloned into plasmids; p67-BHO.6 contained the msr-msd region and pRT-67 contained the RT gene under the lpp-lac promoter-operator. msDNA-Ec67 was produced only when cells were transformed with both plasmids. In addition, msDNA-Ec67 was synthesized in a cell-free system using total RNA prepared from cells harboring plasmid p67-BHO.6 and purified Ec67-RT. Using this cell-free system, the priming reaction, during initiation of DNA synthesis, was demonstrated to be a specific template-directed event; only dTTP was incorporated into a 132-base precursor RNA yielding a 133-base compound. This specific dT addition could be altered to dA or dC by simply substituting the 118th A residue of the putative msr-msd transcript with a T or G residue. The priming reaction was blocked when A was substituted for G at the 15th residue of the precursor RNA transcript, which corresponds to the branched rG residue in msDNA. DNA chain elongation could be terminated by adding ddNTP in the cell-free system, forming a sequence ladder. The DNA sequence determined from this ladder completely agreed with the msDNA sequence. The RT extension reaction was completely blocked when the RNA preparation was treated with RNase A but not when the preparation was treated with DNase. This clearly demonstrates that RNA but not DNA is responsible for the msDNA production. A part of the fully extended cell-free product contained a 13-base RNA strand resistant to RNase A, which is consistent with the previously proposed model. In this model, the 5'-end sequence of the msr-msd transcript (a2; bases 1-13) forms a duplex with the 3'-end sequence (a1) of the same transcript, thus serving as a primer, as well as a template for msDNA synthesis by RT. Our results are inconsistent with a model recently proposed by Lease and Yee (Lease, R. A., and Yee, T. (1991) J. Biol. Chem. 266, 14497-14503).
Base Sequence↗
In vivo production of a stable single-stranded cDNA in Saccharomyces cerevisiae by means of a bacterial retron.
Gram-negative bacteria such as Myxococcus xanthus, Stigmatella aurantiaca, and Escherichia coli contain retroelements called retrons. Retrons consist of the msr-msd region and the gene for reverse transcriptase (RT), which are essential for the production of the branched RNA-linked ms-DNA (multicopy single-stranded DNA). In this study, we attempted to produce msDNA in the yeast Saccharomyces cerevisiae. Retron Ec67 from E. coli, which is responsible for the production of msDNA-Ec67, was cloned under the GAL10 promoter in a 2-microns-based plasmid. msDNA thus produced was detected by extending the 3' end of the msDNA by avian myeloblastosis virus RT. This yielded a main product of 117 nucleotides. Treatment of this product with RNase A resulted in a DNA of 105 nucleotides. These results are in good agreement with the structure of msDNA-Ec67. The production of msDNA-Ec67 was further confirmed by Southern blot hybridization. The msDNA production was dependent upon the bacterial RT gene in the clone and was increased severalfold when the RT gene of retron Ec67 was placed in front of the msr-msd region. The potential of msDNA as a eukaryotic vector producing a stable single-stranded DNA as well as RNA is discussed.
Base Sequence↗
In vivo duplication of genetic elements by the formation of stem-loop DNA without an RNA intermediate.
Gene duplication through cDNA synthesis by reverse transcriptase is believed to have played an important role in the diversification of genomes during evolution. Here, we demonstrate that a genomic DNA sequence can be duplicated in vivo as a result of template switching. When an inverted repeat (IR) structure was inserted in a site downstream from a ColE1 plasmid origin of DNA replication, transformation of Escherichia coli cells with this plasmid resulted in the production of a new DNA fragment encompassing the region from the origin to the center of the IR structure. The structure of this DNA molecule is composed of a long stem-loop formed by a single-stranded DNA, in which the loop is formed by the IR structure. The DNA fragment is designated slDNA, for stem-loop DNA. The experiments in this study suggest that during DNA replication, template switching at the stem-loop structure formed by the IR structure gives rise to slDNA utilizing the nascent DNA strand or the parental strand as a template. The mechanistic implications of slDNA synthesis, and its possible roles in genome evolution, are discussed.
Base Sequence↗
A positive residue in the hydrophobic core of the Escherichia coli lipoprotein signal peptide suppresses the secretion defect caused by an acidic amino terminus.
The signal peptide of secretory proteins requires a basic amino terminus followed by a stretch of hydrophobic residues to effect efficient translocation of precursor proteins. Replacement of the positively charged amino-terminal residues of prolipoprotein by acidic amino acids decreased the rate of precursor translocation (Inouye, S., Soberon, X., Franceschini, T., Nakamura, K., Itakura, K., and Inouye, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3438-3441; Vlasuk, G. P., Inouye, S., Ito, H., Itakura, K., and Inouye, M. (1983) J. Biol. Chem. 258, 7141-7148). We demonstrate here that an arginine residue, but not an aspartate, when localized at position 9 of the hydrophobic region of the lipoprotein signal peptide, is able to suppress intramolecularly the processing defect caused by an acidic amino terminus. Furthermore, when present at position 14 of the signal peptide, this positive residue, but not aspartate, was able to support efficient translocation of unmodified prolipoprotein. This demonstrates that a positive residue can restore the function of a severely defective signal peptide and need not be localized at the amino terminus to do so. Both aspartate and arginine substitution at position 14 of the lipoprotein signal peptide stimulated prolipoprotein synthesis. This effect was position-specific, did not require precursor translocation, and was dominant to the inhibition of synthesis caused by an acidic amino terminus.
Amino Acid Sequence↗
Inhibition of implantation caused by methylmercury and mercuric chloride in mouse embryos in vivo.
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Animals↗
Developmental alteration of serotonin neurons in the raphe nucleus of rats with methylazoxymethanol-induced microcephaly.
Prenatal exposure of pregnant rats to methylazoxymethanol acetate (MAM), an anti-mitotic agent, on day 15 of gestation induces severe microcephaly in the offspring. The present study first investigated a developmental alteration of serotonin (5HT) neurons immunohistochemically in the dorsal and median raphe nuclei in serial sections in both control and microcephalic rats (MAM-rats) at 35 days of age. 5HT-immunoreactive neurons in the MAM-rats were reduced in number and irregularly distributed in the dorsal and median raphe nuclei compared with those in the control. The dendrites of neurons in these nuclei in the MAM-rats were very short and twisted. A follow-up observation on the development of the cerebral cortex at 5, 9 and 28 days of age was performed using Nissl-stained preparations, which revealed a disorganized cell arrangement in the cerebral cortex of the MAM-rats at the very early postnatal period. Furthermore, the distribution of 5HT-immunoreactive fibers into the cerebral cortex was also examined using brains of 28 days of age. In MAM-rats of this age, abnormally tortuous 5HT-immunoreactive fibers were observed in the cerebral cortex. 5HT neurons in the raphe nuclei are known to project their ascending axons widely into the entire cortical area during the 1st postnatal week. Thus, the association of disorganized cortical cell arrangement and the hyperdense and tortuous distribution of 5HT-immunoreactive fibers in the cerebral cortex support the idea of target-dependent secondary degeneration of 5HT neurons in the dorsal and median raphe nuclei of the MAM-rats.
Animals↗
Effects of cycloheximide and actinomycin D on radiation-induced apoptotic cell death in the developing mouse cerebellum.
Effects of cycloheximide and actinomycin D on radiation-induced cell death in the external granular layer (EGL) of the cerebellum were studied in vivo. Newborn mice were exposed to 0.24 Gy gamma-radiation, and dying cells which exhibited pyknosis of nuclei in the EGL were examined at various post-irradiation periods. The number of pyknotic cells began to increase 3 h after irradiation, reached a peak incidence at 6 h, and then gradually fell to the sham-irradiated level by 18 h. When pups were injected with cycloheximide 1 h after irradiation, cell death was suppressed for 6 h, but a peak mortality as high as in the case of radiation alone was attained at 15 h after irradiation. When pups were treated with cycloheximide twice, at 1 and 6 h after irradiation, cell death did not occur for 15 h, but then the incidence rose to a level similar to that after irradiation alone. These findings showed that radiation-induced cell death in the EGL is suppressed by cycloheximide until the chemical is metabolized. Hence, death is by apoptosis which is known to require macromolecular synthesis, and the 'signal' for apoptosis in the cell persists for at least 15 h after irradiation. On the other hand, actinomycin D injected immediately before or after irradiation did not affect the initiation of cell death; actinomycin D alone induced cell death.
Animals↗
The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis.
Subtilisin from a wide variety of Bacillus species has been extensively investigated as a promising target for protein engineering. In this study, we analyzed the substrate specificity of B. subtilis subtilisin E based on the structure of a new alkaline elastase produced by the alkalophilic Bacillus strain Ya-B, which has very high elastolytic activity. Despite the high homology of the primary sequences of both enzymes (54% identical), alkaline elastase was found to lack four consecutive amino acids which, in subtilisin, have been shown by X-ray analysis to lie close to the P1 binding cleft. To examine the influence of such a deletion in subtilisin on its substrate specificity, we constructed several mutants missing four amino acids by site-directed mutagenesis. When assayed with synthetic peptides, elastin and casein as substrates, a mutant lacking Ser161-Thr162-Ser163-Thr164 showed considerably lower specific activity toward the substrates for subtilisin, and its substrate specificity approached that of alkaline elastase. The results indicate that the deletion in subtilisin E influences the catalytic efficiency as well as the P1 specificity, and that this region is, in part, responsible for the difference in specificity between the two enzymes.
Amino Acid Sequence↗
Retron-Ec107 is inserted into the Escherichia coli genome by replacing a palindromic 34bp intergenic sequence.
Some natural isolates of Escherichia coli have been shown to produce a unique branched RNA-linked single-stranded DNA called msDNA. These bacteria contain a retro-element called retron consisting of the msr-msd region and the gene for reverse transcriptase (RT). All three E. coli retrons characterized to date have been shown to be integrated into a prophage or to be associated with phage-related genes. In this report, we identified a new msDNA from an E. coli wild strain. Using the msDNA as a probe, the retron for the msDNA was cloned and its DNA sequence was determined. The retron was found to consist of a 1.3kb DNA fragment, making it the smallest retron isolated to date. The msDNA produced from the retron consists of a 107 base single-stranded DNA, which is considered to be branched out from the 18th G residue of a 75-base RNA molecule by a 2',5'-phosphodiester linkage. Thus, the msDNA and the retron were designated msDNA-Ec107 and retron-Ec107, respectively. Most significantly, retron-Ec107 was inserted into the E. coli genome by replacing a 34bp intergenic sequence between the pyrE and ttk genes located at 82 min on the E. coli chromosome. Interestingly, the retron contains palindromic structures at both ends and the E. coli 34bp intergenic sequence also contains a 10bp inverted repeat structure. These palindromic structures might have played a role in the integration of retron-Ec107 into the E. coli genome.
Amino Acid Sequence↗
Sequence diversity of the 1.3 kb retron (retron-Ec107) among three distinct phylogenetic groups of Escherichia coli.
In the preceding paper, we showed that a new 1.3 kb retron (retron-Ec107) in Escherichia coli is responsible for the biosynthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA-Ec107). Here, we show that this retron occurs in strains from different branches, A, B1, and D of a well-defined phylogenetic tree of a collection of wild E. coli. Sequence comparisons of the retrons from these three branches were carried out. Sequence homology was well conserved among the strains within the same branch and the retron sequence from branch A was exactly the same with that from branch D, while there were 18 base substitutions between the retrons from branch B1 and A or D, resulting in seven amino acid substitutions in reverse transcriptase. No substitutions were found in the msDNA- and msdRNA-coding regions, and there was no difference in the ability of msDNA production between them. These results suggest that the retron has probably been integrated into at least one of the three branches at an early stage of evolution and subsequently transferred to the other two branches, and also that the msDNA-producing system has been conserved during evolution with some mutations in the retron.
Base Sequence↗
Overproduction of a selenocysteine-containing polypeptide in Escherichia coli: the fdhF gene product.
The fdhF gene of Escherichia coli codes for the selenocysteine-including protein subunit of formate dehydrogenase H. The protein subunit consists of 715 amino acid residues containing a single selenocysteine residue at position 140 which is encoded by a UGA codon. The decoding of this opal termination codon occurs under anaerobic growth conditions by means of a specific tRNA, i.e. the selC gene product. The ability of E. coli cells to overproduce a selenopolypeptide was examined using the fdhF gene as a model system. Surprisingly, E. coli was able to synthesize the fdhF gene product at the level of approximately 12% of the total cellular protein. This was achieved by cloning fdhF in a multicopy plasmid together with a synthetic selC gene under the Ipp promoter. FdhF production was absolutely dependent upon the addition of selenium to the culture medium and was almost completely blocked in the presence of oxygen. The product was specifically labelled with 75Se, proving that it consisted of a selenoprotein. The product was purified to homogeneity and shown to exhibit the catalytic properties characteristic of formate dehydrogenase H.
Bacterial Proteins↗
The nlpA lipoprotein gene is located near the selC tRNA gene on the Escherichia coli chromosome.
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Bacterial Proteins↗