Messenger RNA capping enzymes from eukaryotic cells.
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Animals↗
Biomedical subjects
Y Kaziro
Publications and source records attributed to Y Kaziro.
Occurrence in Saccharomyces cerevisiae of a gene homologous to the cDNA coding for the alpha subunit of mammalian G proteins.
From cross-hybridization studies with cDNAs that code for the alpha subunits of rat brain guanine nucleotide-binding regulatory (G) proteins, we have isolated a gene from yeast Saccharomyces cerevisiae encoding an amino acid sequence that is highly homologous to the alpha subunit of the G protein that mediates inhibition of adenylate cyclase (Gi alpha) from rat brain. The gene, tentatively designated as GPA1, contains a contiguous, single open reading frame of 1416 nucleotides that codes for a protein of 472 amino acids with a calculated Mr of 54,075. The predicted amino acid sequence of the protein encoded by the GPA1 gene (tentatively designated as G protein 1 alpha or GP1 alpha) is remarkably homologous to the amino acid sequence of rat brain Gi alpha and the alpha subunit of the G protein of unknown function (Go alpha); the primary structure of the sites for GTP hydrolysis as well as GTP interaction are nearly identical. The main difference in the molecular sizes of yeast GP1 alpha (472 amino acids) and rat brain Gi alpha (355 amino acids) is due to the presence of a stretch of 110 extra amino acid residues in yeast GP1 alpha, which are inserted near the NH2-terminal one-third of mammalian Gi alpha. From blot-hybridization analysis, the size of the GP1 alpha mRNA was estimated as 1.7 kilobases.
Amino Acid Sequence↗
Induction of neurite formation in PC12 cells by microinjection of proto-oncogenic Ha-ras protein preincubated with guanosine-5'-O-(3-thiotriphosphate).
Rat pheochromocytoma (PC12) cells differentiate to neuronal cells in response to nerve growth factor. It has been shown that microinjection of oncogenic but not proto-oncogenic p21 protein induces morphological differentiation in PC12 cells (D. Bar-Sagi and J. R. Feramisco, Cell 42:841-848, 1985). In this paper we describe a recombinant human proto-oncogenic Ha-ras protein which can effectively induce neurite extension of PC12 cells when microinjected as a complex with guanosine-5'-O-(3-thiotriphosphate). The protein was found to be less effective when complexed with GTP. On the other hand, an oncogenic ras protein coinjected with guanosine-5'-O-(2-thiodiphosphate) was entirely inactive. These results indicate that the binary p21-GTP complex, but not the p21-GDP complex, is effective in inducing differentiation in PC12 cells, irrespective of the oncogenic or the proto-oncogenic protein.
Adrenal Gland Neoplasms↗
Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange.
The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.
Antibodies, Monoclonal↗
Role of a ras homolog in the life cycle of Schizosaccharomyces pombe.
We have analyzed the function of the only ras homolog in S. pombe detectable by Southern blotting, ras1, which is homologous to mammalian ras genes and has been cloned. We have disrupted the ras1 gene and have replaced it with ras1Val17, which corresponds to a transforming variant of mammalian ras. Loss of ras1 activity by disruption results in the complete inability to mate. The cell body of a ras1- strain is extensively deformed, and a ras1-/ras1- diploid sporulates very poorly. Unlike RAS1 and RAS2 of S. cerevisiae, ras1 of S. pombe appears to have no effect on adenylate cyclase activity. This suggests that the target enzymes presumably modulated by ras proteins in signal transduction are not the same for all organisms.
Adenylyl Cyclases↗
Mating pheromone-like diffusible factor released by Schizosaccharomyces pombe.
We demonstrate that a diffusible factor is secreted by h cells of the fission yeast Schizosaccharomyces pombe, whose mating pheromones have not been described. This factor, tentatively named the h-factor, affects hS. pombe cells and induces their elongation under nitrogen-depleted conditions. Circumstantial evidence suggests its physiological significance in the mating process. Despite their sterility, hras1 cells secrete this factor. However, hras1 cells have apparently lost the ability to respond to it. This may suggest that the gene product of S. pombe ras1, a homologue of mammalian ras oncogenes, is involved in the mechanism for responding to mating pheromones.
Journal Article↗
Structure of the two genes coding for polypeptide chain elongation factor 1 alpha (EF-1 alpha) from Saccharomyces cerevisiae.
Polypeptide chain elongation factor 1 alpha (EF-1 alpha) of Saccharomyces cerevisiae is encoded by two distinct genes designated EF1 alpha A and EF1 alpha B [Nagata et al., EMBO J. 3 (1984) 1825-1830]. Both genes were cloned, and their nucleotide (nt) sequences were determined [see also Schirmaier and Philippsen, EMBO J. 3 (1984) 3311-3315, and Cottrelle et al., J. Biol. Chem. 260 (1985) 3090-3096]. They contain an open reading frame of 1374 nt coding for an identical protein of 458 amino acid residues, although their nt sequences differed at two positions. In this paper, we determined their 5'- and 3'-flanking sequences which were considerably different each other. From the S1 nuclease mapping of mRNA, both genes are found to be expressed almost to the same extent in exponentially growing cells. The transcription start points for EF1 alpha A and EF1 alpha B mRNAs were precisely located by primer extension procedure at 32 and 23 nt upstream of the start codons, respectively. The sequence which commonly exists in the 5'-flanking regions of ribosomal protein genes of S. cerevisiae was also present in the two EF1 alpha genes.
Amino Acid Sequence↗
Isolation and characterization of the cDNA for murine granulocyte colony-stimulating factor.
A cDNA sequence coding for murine granulocyte colony-stimulating factor (G-CSF) has been isolated from a cDNA library prepared with mRNA derived from murine fibrosarcoma NFSA cells, which produce G-CSF constitutively. Identification of murine G-CSF cDNA was based on the cross-hybridization with human G-CSF cDNA under a low-stringency condition. The cDNA can encode a polypeptide consisting of a 30-amino acid signal sequence, followed by a mature G-CSF sequence of 178 amino acids with a calculated Mr of 19,061. The nucleotide sequence and the deduced amino acid sequence of murine G-CSF cDNA were 69.3% and 72.6% homologous, respectively, to the corresponding sequences of human G-CSF cDNA. The murine G-CSF cDNA, when introduced into monkey COS cells under the simian virus 40 promoter, could direct the synthesis of a protein that can stimulate the granulocyte colony formation from mouse bone marrow cells and support the proliferation of murine NFS-60 myeloid leukemia cells.
Amino Acid Sequence↗
Molecular cloning and sequence analysis of a ras gene from Schizosaccharomyces pombe.
We have cloned a ras gene homologue from fission yeast Schizosaccharomyces pombe and determined its nucleotide sequence. A putative coding sequence for 219 amino acids was found. The sequence contained one set of splicing signals: GTAAGT for a donor sequence, ACTAA for a unique sequence found in introns of yeast genes and TAG for an acceptor sequence, indicating the existence of an intron. The amino-terminal one third of the predicted S. pombe ras protein was nearly perfectly homologous and the next one third moderately homologous to those of mammalian ras proteins. The carboxy-terminal one third showed no homology but terminated with a short conserved sequence Cys-X-X-Z (X being a hydrophobic amino acid) as in other ras proteins. The result of Southern analysis of S. pombe DNA under nonstringent hybridization conditions using our clone as a probe indicated that no other closely related gene may be present in the S. pombe genome. The transcript of this gene could be detected by Northern analysis.
Amino Acid Sequence↗
Regulation of the expression of the tufB operon: DNA sequences directly involved in the stringent control.
We have located the DNA sequence involved in the stringent control of the Escherichia coli tufB operon. Various deletion and insertion mutants of the promoter locus were constructed by in vitro mutagenesis, and their response to guanosine-5'-diphosphate-3'-diphosphate (ppGpp) was examined in a cell-free transcription system consisting of purified RNA polymerase holoenzyme. The nucleotide sequence (GpCpGpC) from positions -7 to -4 (designating the initiation site of mRNA as position +1) is responsible for the selective inhibition by ppGpp of tufB transcription. Point mutations were then constructed in which each one of the above four nucleotides was replaced by an A or T residue and tested for their response to ppGpp in the in vitro transcription system. The results indicated that the alteration of any nucleotide in the GpCpGpC sequence leads to the loss of the stringent response.
Base Sequence↗
[Molecular mechanism of stringent control].
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Base Sequence↗
Messenger RNA guanlyltransferase from Saccharomyces cerevisiae. II. Catalytic properties.
Highly purified mRNA-capping enzyme from Saccharomyces cerevisiae catalyzes (a) removal of the gamma-phosphoryl group from the 5'-end of the newly formed mRNA and (b) guanylylation of the resulting diphosphoryl end. Characteristics of the two reactions catalyzed by this enzyme are studied. Guanylyltransferase is most active at pH 7.0 in the presence of 3 mM Mg2+, and utilizes GTP as a guanylyl donor with an apparent Km of 5 microM, and ppGCC (A2, U2, G)n as a guanylyl acceptor with two Km values of 0.5 and 4 microM. It catalyzes GTP-PPi exchange in the absence of the acceptor RNA, and forms a covalent enzyme-GMP intermediate having Mr = 45,000 in sodium dodecyl sulfate gel electrophoresis. RNAs with 5'-diphosphoryl as well as 5'-triphosphoryl ends are capped, while mononucleotides such as GDP and ppGp are inert. Since guanylyltransferase can utilize ppGpC and ppGpCpC as acceptors, the presence of at least one phosphodiester bond seems to be sufficient for the acceptor activity. However, oligonucleotides of longer chain length are preferred. RNA 5'-triphosphatase associated with the purified enzyme requires Mg2+ and exhibits a broad pH optimum from 6.5 to 8.5, and an apparent Km value for pppA-terminated poly(A) is 1.4 microM. The enzyme is specific for the gamma-phosphoryl group at the 5'-terminus of RNA and does not hydrolyze ATP. It can hydrolyze the gamma-phosphoryl group of pppGp, but the RNA substrates with longer chain length are preferred.
Guanosine Monophosphate↗
Messenger RNA guanylyltransferase from Saccharomyces cerevisiae. I. Purification and subunit structure.
GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of the GMP moiety from GTP to the 5' end of the RNA to form a cap structure (G(5')pppN-), has been purified to an apparent homogeneity from Saccharomyces cerevisiae. The mRNA 5'-triphosphatase activity hydrolyzing the gamma-phosphoryl group from pppN-RNA was co-purified with mRNA guanylyltransferase activity through column chromatographies on CM-Sephadex and poly(U)-Sepharose, and centrifugation through glycerol gradients, suggesting that these two activities are physically associated. An 820,w value of 7.3, and Mr = 140,000 were estimated from the sedimentation behavior in glycerol gradients. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two major polypeptides, Mr = 45,000 (alpha) and 39,000 (beta), were detected with the purified enzyme preparation. Their molar ratios were close to unity when estimated by the relative density of silver staining. These results suggest that the yeast mRNA-capping enzyme is an oligomeric protein which may consist of two alpha and two beta chains (alpha 2 beta 2).
Chromatography, Affinity↗
Limited tryptic digestion of messenger RNA capping enzyme from Artemia salina. Isolation of domains for guanylyltransferase and RNA 5'-triphosphatase.
The partially purified preparation of messenger RNA guanylyltransferase from Artemia salina contains, as in the case of the rat liver enzyme (Yagi, Y., Mizumoto, K., and Kaziro, Y. (1983) EMBO J. 2, 611-615), the RNA 5'-triphosphatase activity which specifically removes the gamma-phosphoryl group from the 5'-triphosphoryl end of the newly synthesized mRNA molecule. The enzyme consists of a single polypeptide chain of Mr = 73,000 and forma a covalent enzyme-GMP complex as an intermediate for the guanylyltransferase reaction. Upon limited hydrolysis with trypsin, the enzyme-[32P]GMP complex is converted to a smaller 32P-containing fragment of Mr = 44,000. When the free enzyme, not complexed with GMP, is digested with trypsin under the same condition as above, the digests retain almost full activities of both guanylyltransferase and RNA 5'-triphosphatase and can form an enzyme-[32P]GMP complex of the size of Mr = 44,000 on incubation with [alpha-32P]GTP. Functional domains harboring the activities of guanylyltransferase and RNA 5'-triphosphatase are separated by gel filtration on a Sephacryl S-200 column at positions corresponding to Mr = 44,000 and 20,000, respectively. They can be separated completely from each other by CM-Sephadex column chromatography. While the native, undigested enzyme can transfer the GMP moiety to mRNA molecules with either triphosphoryl (pppN-) or diphosphoryl (ppN-)5'terminal, the purified Mr = 44,000 domain with the guanylyltransferase activity can utilize only the latter as an acceptor.
Acid Anhydride Hydrolases↗
Structural and functional studies of the dnaB protein using limited proteolysis. Characterization of domains for DNA-dependent ATP hydrolysis and for protein association in the primosome.
Two separable structural domains were identified in the Escherichia coli dnaB protein (Mr = 52,000) by partial proteolytic cleavage under nondenaturing conditions. The hydrolysis of dnaB protein by trypsin proceeded in two distinct stages in the presence of ATP or ADP. In the first stage, 14 amino acid residues at the NH2-terminal end were removed and dnaB protein was converted into a fragment with a molecular weight of 50,000 (Fragment I). Fragment I retained about 60% of the original activity in priming DNA replication and was fully active in DNA-dependent ATPase activity. In the second stage, Fragment I was further cleaved into two separable polypeptides with molecular weights of 33,000 (Fragment II) and 12,000 (Fragment III), respectively. Fragment II, as a hexamer, retained DNA-dependent ATPase activity comparable to the intact protein but was totally inactive in priming DNA replication. No known activity of dnaB protein was detected in Fragment III alone. NH2 termini of Fragments I and III and COOH termini of dnaB protein and Fragment II were identical indicating that Fragments III and II were located at the NH2 and COOH termini of Fragment I, respectively. These results indicate that dnaB protein is composed of at least two distinct domains. 1) Fragment III, the rigid domain, is essential for protein interaction, i.e. association with dnaC protein and primase in priming DNA replication in the primosome. 2) A 14-amino acid residue fragment, at the NH2-terminal end adjacent to Fragment III, probably required to stabilize the protein interaction involved in priming DNA replication. 3) Fragment II, the flexible COOH-terminal domain, contains the active sites for DNA binding, ATP binding, and protein oligomerization. Fragment II is cleaved by trypsin at many sites in the absence of ATP or ADP ligands. The rate of conversion of Fragment I into the yield of Fragments II and III was decreased approximately by 2 orders of magnitude by changing the ligand from ADP to the nonhydrolyzed ATP analog, adenosine 5'-O-(3-thiotriphosphate). These results indicate that the conformation of the COOH-terminal domain in the dnaB protein is stabilized by ATP or ADP. Such a nucleotide-induced conformational change was also demonstrated by circular dichroism spectroscopy. Moreover, the data suggest that the conformation of the dnaB protein complexed with adenosine 5'-O-(3-thiotriphosphate) is different from that complexed with ADP.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine Triphosphate↗
Nucleotide sequence of dnaB and the primary structure of the dnaB protein from Escherichia coli.
We have determined the nucleotide sequence of the dnaB gene and the primary structure of the dnaB protein of Escherichia coli (Arai, K., Yasuda, S., and Kornberg, A. (1981) J. Biol. Chem. 256, 5247-5252). The coding region for the dnaB protein is 1413 base pairs followed by double stop codons and preceded by a possible promoter sequence. The dnaB gene lacks a typical Shine-Dalgarno sequence. The primary structure deduced from the DNA sequence is consistent with the protein chemical data. The dnaB protein contains 470 amino acid residues and has a calculated molecular weight of 52,265. In the mature protein, the initiator methionine residue is removed in vivo leaving alanine as the NH2-terminal residue. Based on the amino acid sequence, we predict that the dnaB protein may be composed of two domains. A hydrophilic NH2-terminal region (residues 1-20) is followed by a compact domain and a possible hinge region (residues 21-172) consisting primarily of alpha-helix. The sites of facile tryptic cleavage are at the arginine residues at 14 and 171. The DNA-dependent ATPase domain (residues 172-470) is located at the COOH-terminal end of the protein.
Amino Acid Sequence↗
Polypeptide chain elongation factor 1 alpha (EF-1 alpha) from yeast: nucleotide sequence of one of the two genes for EF-1 alpha from Saccharomyces cerevisiae.
Messenger RNA for yeast cytosolic polypeptide chain elongation factor 1 alpha (EF-1 alpha) was partially purified from Saccharomyces cerevisiae. Double-stranded complementary DNA (cDNA) was synthesized and cloned in Escherichia coli with pBR327 as a vector. Recombinant plasmid carrying yEF-1 alpha cDNA was identified by cross-hybridization with the E. coli tufB gene and the yeast mitochondrial EF-Tu gene (tufM) under non-stringent conditions. A yeast gene library was then screened with the EF-1 alpha cDNA and several clones containing the chromosomal gene for EF-1 alpha were isolated. Restriction analysis of DNA fragments of these clones as well as the Southern hybridization of yeast genomic DNA with labelled EF-1 alpha cDNA indicated that there are two EF-1 alpha genes in S. cerevisiae. The nucleotide sequence of one of the two EF-1 alpha genes (designated as EF1 alpha A) was established together with its 5'- and 3'-flanking sequences. The sequence contained 1374 nucleotides coding for a protein of 458 amino acids with a calculated mol. wt. of 50 300. The derived amino acid sequence showed homologies of 31% and 32% with yeast mitochondrial EF-Tu and E. coli EF-Tu, respectively.
Amino Acid Sequence↗
Molecular cloning and nucleotide sequence of cDNA for rat ornithine carbamoyltransferase precursor.
Messenger RNA of rat ornithine carbamoyltransferase (EC 2.1.3.3), a mitochondrial matrix enzyme, was enriched by immunoprecipitation of rat liver free polysomes, and recombinant plasmids were prepared from the enriched mRNA by a vector-primer method. The cDNA clones for ornithine carbamoyltransferase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pOTC-1, contained a 1.6-kilobase insert and hybridized to a mRNA of approximately equal to 1.8 kilobases in rat liver. The cDNA clone was subjected to nucleotide sequence analysis. The deduced amino acid sequence indicates that the ornithine carbamoyltransferase precursor consists of the mature enzyme of 322 amino acid residues and an NH2-terminal peptide extension (presequence) of 32 amino acid residues. The presequence contains 8 basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The amino acid sequence of the rat ornithine carbamoyltransferase was compared with the recently reported sequence of the human enzyme [Horwich, A. L., Fenton, W. A., Williams, K. R., Kalousek, F., Kraus, J. P., Doolittle, R. F., Konigsberg, W. & Rosenberg, L. E. (1984) Science 224, 1068-1074]. The sequences of the mature enzyme portion are 93% identical, whereas those of the presequences are 69% identical. There are two highly conserved segments in the presequences of the rat and human enzymes. One of the two conserved segments is significantly similar to a segment of the presequence of yeast mitochondrial elongation factor EF-Tu. These results suggest that the homologous segments are important for the proteins that are synthesized in the cytosol to be transported into the mitochondrial matrix.
Amino Acid Sequence↗