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Biomedical subjects

M Inouye

Publications and source records attributed to M Inouye.

At least 199 records · Page 11Linked to original sources
PubMed1994

The OmpR protein of Escherichia coli binds to sites in the ompF promoter region in a hierarchical manner determined by its degree of phosphorylation.

In Escherichia coli the ompF gene encodes a major outer membrane porin protein that is differentially regulated by the OmpR protein. OmpR acts as a positive as well as a negative regulator of ompF expression by binding to DNA sequences in the ompF promoter region. The DNA binding activity of OmpR is itself regulated by phosphorylation through the kinase protein EnvZ. Phosphorylation is believed to change the function of OmpR from an activator to a repressor molecule. By using purified OmpR and various regions of the ompF promoter we show that phosphorylation causes binding of OmpR to a DNA region between the -40 to -100 region of the ompF promoter previously shown to be important for ompF expression. As the amount of OmpR-phosphate increases, a binding site located at a further upstream -360 to -380 region was occupied. This latter site has been reported to be important for ompF repression. Further experiments indicate that the -70 to -100 region is a high affinity site, while the -45 to -60 and -360 to -380 regions are low affinity sites. We also provide evidence that OmpR binding at the -360 to -380 region requires previous binding at downstream sequences, which is indicative of long range interactions between OmpR molecules. We interpret our results in terms of a model for ompF regulation involving hierarchical binding by phosphorylated OmpR and potential DNA looping.

Bacterial Outer Membrane Proteins↗
PubMed1994

Cloning and sequences of two macrolide-resistance-encoding genes from mycinamicin-producing Micromonospora griseorubida.

Two macrolide-resistance determinants were cloned from a mycinamicin (Mm)-producing Micromonospora griseorubida strain in Streptomyces lividans and Streptomyces parvulus. One of the cloned genes, designated myrA, was cloned as a gene which conferred strong resistance to Mm and tylosin (Ty), but not to erythromycin (Er) or josamycin (Jm) on S. lividans. Another gene, named myrB, was cloned as an ErR-encoding gene which conferred MLS resistance (to macrolide, lincosamide and streptogramine B antibiotics) on S. parvulus. Both myrA and myrB were sequenced and the corresponding ORFs were determined. The deduced amino acid (aa) sequence of myrA showed no similarity to proteins in the available databases, suggesting that an unknown mechanism of macrolide resistance is exerted by the MyrA protein. The deduced aa sequence of myrB exhibited high similarity to 23S rRNA methyltransferases (MTases), such as ErmE and CarB, from a variety of microorganisms.

Amino Acid Sequence↗
PubMed1994

A gene encoding mycinamicin III O-methyltransferase from Micromonospora griseorubida.

A DNA fragment of 42 kb encompassing one of the mycinamicin II (Mm)-resistance-encoding genes, myrB, from a Mm-producing strain, Micromonospora griseorubida, was cloned in Escherichia coli using the cosmid vector pJB8. Nucleotide sequencing of the neighboring region of myrB and a computer-aided analysis of the sequence predicted the presence of an open reading frame (ORF) with 254 amino acids which showed great similarity to the macrocin O-methyltransferase (tylF gene product) in tylosin (Ty)-producing Streptomyces fradiae. When a 1.0-kb AluI fragment containing the complete ORF was fused to the lacZ promoter in the correct orientation and expressed in E. coli, a mycinamicin III (MIII) O-methyltransferase (MOMT) activity was detected only upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). All these data indicate that this ORF codes for the structural gene of MOMT, and it is designated mycF.

Amino Acid Sequence↗
PubMed1994

Identification of cis- and trans-acting elements involved in the expression of cold shock-inducible TIP1 gene of yeast Saccharomyces cerevisiae.

Northern blot hybridization analysis of a series of 5' end, 3' end and internal deletions has revealed that at least four different regions are involved in the regulation of the expression of TIP1, a cold shock-inducible gene of Saccharomyces cerevisiae. One of these four regions has negative effect on the expression of the TIP1 gene, while the others are responsible for the activation and cold shock-induction of the gene. A fragment involved in the cold-shock induction of TIP1 was used as a probe in gel retardation assays to identify the cold shock-factor. The cold shock-factor could be detected in cells grown at 30 degrees C as well as 10 degrees C, but both the amount of the factor and its affinity to DNA were found to increase 2-3-fold after cold shock. In addition, another factor was found to bind just upstream of the cold shock element, in a region where a transcriptional activator was predicted to function by Northern blot hybridization analysis. The amount of this activating factor and its affinity for DNA was not affected by temperature. Implications of our data on possible mechanisms of transcriptional regulation of the TIP1 gene by cold shock are discussed.

Base Sequence↗
PubMed1994

Specificity of priming reaction of HIV-1 reverse transcriptase, 2'-OH or 3'-OH.

It has not been unambiguously demonstrated whether the priming reaction of human immunodeficiency virus, type 1 (HIV-1) cDNA synthesis initiates with either the 2'-OH or 3'-OH group of the 3'-terminal adenosine residue of tRNA(Lys-3). In this report, we synthesized tRNA(Lys-3) of which the 3'-terminal adenosine residue lacks either a 2'-OH or 3'-OH. These tRNA molecules were used for the HIV-1 cDNA-priming reaction in a cell-free system consisting of a 141-base RNA template and purified HIV-1 reverse transcriptase. It was found that under the conditions used, the tRNA containing the 2'-deoxyadenosine was able to initiate the cDNA synthesis, while the tRNA with the 3'-deoxyadenosine was not. The results show that retroviral reverse transcriptase specifically primes cDNA synthesis from the 3'-OH group. This is in contrast to bacterial reverse transcriptase, which initiates cDNA synthesis from the 2'-OH group of an internal guanosine residue of a template RNA.

Base Sequence↗
PubMed1994

Autoprocessing of prothiolsubtilisin E in which active-site serine 221 is altered to cysteine.

Subtilisin, an extracellular serine protease from Bacillus subtilis, requires the amino-terminal propeptide of 77 amino acid residues for the formation of the active enzyme. The propeptide is cleaved upon completion of folding. Serine 221 at the active center was substituted with cysteine, and the mutant enzyme (prothiolsubtilisin) was expressed in Escherichia coli under the control of a T7 promoter. Prothiosubtilisin, which was produced as inclusion bodies, was dissolved in 6 M guanidine HCl and purified to near homogeneity in the presence of 5 M urea. The purified protein was renatured by stepwise dialysis. In spite of the mutation at the active center, the propeptide was found to be autoprocessed with approximately 60-80% efficiency. However, protease activity could not be detected in the final product by the spectrophotometric assay. Moreover, the cleaved propeptide remained tightly bound to thiolsubtilisin without being digested, as evident by SDS-polyacrylamide gel electrophoresis. The amino-terminal sequence of the processed thiolsubtilisin was determined and proved that the propeptide was cleaved at a site identical to that of wild-type prosubtilisin. The processed thiolsubtilisin was also found to contain one free SH group/molecule. These results unambiguously demonstrate that the processing of prosubtilisin occurs by an intramolecular autoprocessing mechanism.

Bacillus subtilis↗
PubMed1994

Identification of the Myxococcus xanthus 59-kDa membrane-associated GTP-binding protein as a proton-translocating ATPase.

Five GTP-binding proteins have been detected in Myxococcus xanthus by photoaffinity cross-linking with azido-GTP [Muñoz-Dorado et al., J. Biol. Chem. 265 (1990a) 2702-2706]. One of them, the 59-kDa membrane-associated GTP-binding protein, has been purified. The N-terminal sequence of a 10-kDa fragment from the protease V8 digestion of the purified protein has been determined and degenerate oligodeoxyribonucleotides based on that sequence have been used to isolate and clone the gene that encodes the GTP-binding protein. The gene was sequenced and further analysis of the sequence revealed that the protein encoded by this gene shows very high homology with the alpha subunit of proton-translocating ATPases.

Amino Acid Sequence↗
PubMed1994

Production of single-stranded DNA in mammalian cells by means of a bacterial retron.

msDNA-Ec67, a peculiar multicopy single-stranded DNA of a specific sequence was produced in NIH3T3 mouse cells. Retron-Ec67, a retroelement from Escherichia coli, was introduced under the T7 polymerase promoter and the non-translated 5'-region of the encephalomyocarditis virus. The construct was then transfected into the NIH3T3 cells constitutively producing T7 RNA polymerase. Forty-eight hours after transfection, msDNA-Ec67 was detected in the cells by means of Southern blot hybridization and reverse transcriptase extension assay. The potential use of bacterial retrons as a vector for single-stranded DNA production in mammalian cells is discussed.

3T3 Cells↗
PubMed1994

A covalently trapped folding intermediate of subtilisin E: spontaneous dimerization of a prosubtilisin E Ser49Cys mutant in vivo and its autoprocessing in vitro.

The propeptide of subtilisin E (the N-terminal 77 amino acid extension) is required for the proper folding of the nascent mature protein and is also a potent and specific inhibitor of the active enzyme. Previous studies have demonstrated that the propeptide can renature denatured mature sequence either in cis or in trans and can be considered an intramolecular chaperone, since it is not required for activity of the mature enzyme. In this paper it is shown that a prosubtilisin-S49C mutant can be expressed in Escherichia coli either as a monomer or as a disulfide-linked dimer, (prosubtilisin-S49C)2, depending on the vector selected. Interconversion between (prosubtilisin-S49C)2 and prosubtilisin-S49C could be readily achieved by reduction and oxidation in denaturing solutions, such as guanidine hydrochloride or urea. While the monomer can undergo autoprocessing in vitro under refolding conditions, the dimer is trapped in an intermediate state which could not be processed into active enzyme. Remarkably, the autoprocessing of this trapped intermediate could be induced readily upon reduction by dithiothreitol. This disulfide-linked (prosubtilisin-S49C)2 is fairly stable, but does tend to aggregate when the ionic strength of the solution is reduced below 0.1 M. The disulfide-linked (prosubtilisin-S49C)2 has far- and near-UV CD spectra revealing the presence of both secondary and tertiary structures, respectively, similar to those of the active mature monomer. Hence this autoprocessing-competent state appears to be a "late" folding intermediate, arising after the "molten globule" state formed in the absence of the prosequence, that has no discernible tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

The alpha subunit of RNA polymerase specifically inhibits expression of the porin genes ompF and ompC in vivo and in vitro in Escherichia coli.

Overproduction of the alpha subunit of RNA polymerase in Escherichia coli resulted in inhibition of transcription of two osmoregulated porin genes, ompF and ompC, but not of constitutively expressed housekeeping genes. Overproduction of the sigma subunit did not have any inhibitory effects. The specific inhibitory effect of the alpha subunit was also found to depend upon the OmpR protein, the transcriptional activator for ompF and ompC. These results are in general agreement with other biochemical and genetic evidence suggesting that the alpha subunit is the subunit of RNA polymerase that directly interacts with certain transcriptional activators to initiate transcription.

Bacterial Outer Membrane Proteins↗
PubMed1994

The structural and functional organization of intramolecular chaperones: the N-terminal propeptides which mediate protein folding.

A large number of prokaryotic as well as eukaryotic proteins are produced with amino terminal propeptides. These amino-terminal extensions are essential for mediating proper folding of their corresponding proteins and are also termed as intramolecular chaperones. Though these propeptides are highly specific and unique in their function, several common features have been identified and indicate that the overall mechanism by which they function may be very similar.

Amino Acid Sequence↗
PubMed1994

The cold-shock response--a hot topic.

The cold-shock response of Escherichia coli describes a specific pattern of gene expression in response to abrupt shifts to lower temperatures. This pattern includes the induction of cold-shock proteins, synthesis of proteins involved in transcription and translation, and repression of heat-shock proteins. The identified cold-shock proteins are involved in various cellular functions from supercoiling of DNA to initiation of translation. The major cold-shock protein, CspA, has high sequence similarity with three other E. coli proteins--CspB, CspC, and CspD. Using translational lacZ fusions, cspB was found to be cold-shock inducible at the level of transcription like cspA, while cspC and cspD were not. The Csp proteins, which share sequence similarity with other prokaryotic proteins and with the 'cold-shock domain' of eukaryotic Y-box proteins, may have a function in activating transcription or unwinding or masking RNA molecules. Because the cold-shock response can also be induced by the addition of certain inhibitors of translation, it has been proposed that the state of the ribosome is the physiological sensor for the induction. In addition to E. coli, cold-shock proteins have also been found in other prokaryotic and eukaryotic organisms.

Amino Acid Sequence↗
PubMed1994

Reversible topology of a bifunctional transmembrane protein depends upon the charge balance around its transmembrane domain.

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminal OmpA signal sequence was fused with TEM beta-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the beta-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor. In the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal beta-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the amino-terminal nuclease domain across the membrane, leaving the carboxyl terminal beta-lactamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.

Amino Acid Sequence↗
PubMed1994

Family of the major cold-shock protein, CspA (CS7.4), of Escherichia coli, whose members show a high sequence similarity with the eukaryotic Y-box binding proteins.

The cspA is a gene of Escherichia coli, whose expression is specifically induced at low temperatures to a level of 13% of total protein synthesis. The CspA protein consisting of 70 amino acid residues has high sequence similarity with eukaryotic Y-box DNA-binding proteins. We found two independent clones from the Kohara miniset phage collection, which hybridized with a DNA fragment containing cspA. DNA sequencing of these clones confirmed that the two genes are highly homologous to cspA. One designated cspB is mapped at 35 min on the E. coli chromosome and encodes a 71-residue protein with 79% identity to CspA, while the other, cspC, is mapped at 40 min and encodes a 69-residue protein with 70% identity. In addition, a DNA sequence upstream of the clpA gene at 19 min published elsewhere contains an open reading frame for a 74-residue protein with 45% identity to CspA. All csp genes were fused in the coding regions with the lacZ gene, and the expression of beta-galactosidase was examined for these hybrid genes upon cold shock. A similar cold-shock induction to cspA was observed for cspB but not cspC and cspD. These results indicate that E. coli has a family of the cspA gene, some of which are induced by cold shock.

Amino Acid Sequence↗
PubMed1994

Characterization of the autophosphorylation of Era, an essential Escherichia coli GTPase.

Era is an essential protein in Escherichia coli which binds both GTP and GDP and has an intrinsic GTPase activity. Studies on the role of GTP/GDP binding and GTPase activity in an attempt to understand its function lead to the observation that Era is autophosphorylated. The autophosphorylation reaction is specific for GTP and cannot use ATP as a phosphoryl group donor. The reaction velocity is of first order with respect to protein concentration, suggesting an intramolecular mechanism. Autophosphorylation occurs at serine and threonine residues. The major phosphorylated tryptic peptide isolated after autophosphorylation has been identified as ISITSR, from residue 33 to 38. The peptide contains the site of phosphorylation and two potential sites for serine and threonine phosphorylation. Subsequently, both the threonine residue at position 36 and the serine residue at position 37 were altered to alanine. The double mutant Era, but not individual single mutants, was unable to functionally complement the growth of an E. coli strain which cannot produce wild-type Era protein at high temperature. This suggests that either threonine 36 or serine 37 has to exist for the function of Era in vivo. In vivo phosphorylation of Era was also examined by two-dimensional gel electrophoresis. Era has been previously assigned two distinct positions having two different X-Y co-ordinates: one of the spots (H032.0) was identified as phosphorylated Era, indicating that a substantial portion of Era in the cell is indeed phosphorylated. Therefore, Era autophosphorylation is likely to play an important physiological role in the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ.

Chemoreceptor Trg and osmosensor EnvZ of Escherichia coli share a common transmembrane organization but have essentially unrelated primary structures. We created a hybrid gene coding for a protein in which Trg contributed its periplasmic and transmembrane domains as well as a short cytoplasmic segment and EnvZ contributed its cytoplasmic kinase/phosphatase domain. Trz1 transduced recognition of sugar-occupied, ribose-binding protein by its periplasmic domain into activation of its cytoplasmic kinase/phosphatase domain as assessed in vivo by using an ompC-lacZ fusion gene. Functional coupling of sugar-binding protein recognition to kinase/phosphatase activity indicates shared features of intramolecular signalling in the two parent proteins. In combination with previous documentation of transduction of aspartate recognition by an analogous fusion protein created from chemoreceptor Tar and EnvZ, the data indicate a common mechanism of transmembrane signal transduction by chemoreceptors and EnvZ. Signalling through the fusion proteins implies functional interaction between heterologous domains, but the minimal sequence identity among relevant segments of EnvZ, Tar, and Trg indicates that the link does not require extensive, specific interactions among side chains. The few positions of identity in those three sequences cluster in transmembrane segment 1 and the short chemoreceptor sequence in the cytoplasmic part of the hybrid proteins. These regions may be particularly important in physical and functional coupling. The specific cellular conditions necessary to observe ligand-dependent activation of Trz1 can be understood in the context of the importance of phosphatase control in EnvZ signalling and limitations on maximal receptor occupancy in binding protein-mediated recognition.

Amino Acid Sequence↗