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Biomedical subjects

K S Browning

Publications and source records attributed to K S Browning.

At least 37 records · Page 2Linked to original sources
PubMed1996

Mutational analysis of the functional domains of the large subunit of the isozyme form of wheat initiation factor eIF4F.

The isozyme form of plant eukaryotic initiation factor 4F (eIF(iso)4F) contains two subunits: p28, a cap-binding protein, and p86. To identify the functional domains of p86, truncations of the p86 cDNA were made, and the protein was expressed in Escherichia coli and purified. The deletion mutants were tested for the ability to bind the p28 subunit by two methods. In addition, these deletion mutants were evaluated in vitro by the ability to catalyze eIF4A and RNA-dependent ATP hydrolysis and to support polypeptide synthesis. The loss of the ability to bind p28 occurs within the first 90 amino acids of the N terminus and abrogates the ability of p86 to participate in translation initiation and bind to eIF4A, but does not affect ATP hydrolysis. Up to 299 amino acid residues from the C terminus of p86 must be deleted before an effect is observed on the ATP hydrolysis activity. Thus, the p28 binding and ATP hydrolysis activities appear to lie on two separate domains and are functionally uncoupled. In addition, at least a portion of the eIF4A binding domain appears to be in close proximity to the p28 binding domain and is also uncoupled from the ATP hydrolysis activity.

Adenosine Triphosphate↗
PubMed1996

Sequences of two expressed sequence tags (EST) from rice encoding different cap-binding proteins.

Wheat has been shown to have two forms of the cap-binding protein that participate in the initiation of translation. To identify cap-binding proteins from other higher plant species, the expressed sequence tag (EST) database was searched. Several rice ESTs were identified with similarity to both forms of the wheat cap-binding proteins. Two of the rice ESTs were obtained and the cDNA sequences completed. The deduced amino acid sequences of the rice cap-binding proteins are compared to the wheat cap-binding proteins and cap-binding proteins from Saccharomyces cerevisiae, Drosophila melanogaster, Xenopus laevis and human.

Amino Acid Sequence↗
PubMed1996

pH-dependent and ligand induced conformational changes of eucaryotic protein synthesis initiation factor eIF-(iso)4F: a circular dichroism study.

The structural features of wheat germ protein synthesis initiation factor eIF-(iso)4F, which has a cap binding protein as one of its two subunits, are unknown. In this study, circular dichroism (CD) spectra and secondary structure prediction were obtained for eIF-(iso)4F and its two subunits, p28 and p86. The alpha-helix content of eIF-(iso)4F changed from 42% at pH 6.3 to 15% at pH 7.6, the optimum pH for cap binding. The beta-sheet content increased from 14% (pH 6.3) to 38% at pH 7.6. The CD spectra of the two subunits, p28 and p86 were also measured and analyzed. The separated subunits both had a higher alpha-helix content at pH 7.6 than the native protein, giving values of 60% and 34% alpha-helix for p28 and p86, respectively. Binding of the dinucleotide cap analog to p28 reduced the alpha-helix content to approximately 8% with an increase in the beta sheet content from 10% to 37%. The conformational changes in eIF-(iso)4F upon binding with mRNA are dependent on cap or oligonucleotide structure. A conformation consisting of approximately the same alpha-helix and beta-sheet content can be induced by ligands even at non-optimal pH values. This large conformational transition suggests eIF-(iso)4F binds nucleic acids by interaction of a beta-sheet motif and that this conformational transition may have a regulatory role.

Circular Dichroism↗
PubMed1996

Phosphorylation of plant eukaryotic initiation factor-2 by the plant-encoded double-stranded RNA-dependent protein kinase, pPKR, and inhibition of protein synthesis in vitro.

Regulation of protein synthesis by eukaryotic initiation factor-2alpha (eIF-2alpha) phosphorylation is a highly conserved phenomenon in eukaryotes that occurs in response to various stress conditions. Protein kinases capable of phosphorylating eIF-2alpha have been characterized from mammals and yeast. However, the phenomenon of eIF2-alpha-mediated regulation of protein synthesis and the presence of an eIF-2alpha kinase has not been demonstrated in higher plants. We show that plant eIF-2alpha (peIF-2alpha) and mammalian eIF-2alpha (meIF-2alpha) are phosphorylated similarly by both the double-stranded RNA-binding kinase, pPKR, present in plant ribosome salt wash fractions and the meIF-2alpha kinase, PKR. By several criteria, phosphorylation of peIF-2alpha is directly correlated with pPKR protein and autophosphorylation levels. Significantly, pPKR is capable of specifically phosphorylating Ser51 in a synthetic eIF-2alpha peptide, a key characteristic of the eIF-2alpha kinase family. Taken together, these data support the concept that pPKR is a member of the eIF-2alpha kinase family. In addition, the inhibition of brome mosaic virus RNA in vitro translation in wheat germ lysates by the addition of double-stranded RNA, phosphorylated peIF-2alpha, meIF-2alpha, or activated human PKR suggests that plant protein synthesis may be regulated via phosphorylation of eIF-2alpha.

Amino Acid Sequence↗
PubMed1996

The plant translational apparatus.

Protein synthesis in both eukaryotic and prokaryotic cells is a complex process requiring a large number of macromolecules: initiation factors, elongation factors, termination factors, ribosomes, mRNA, amino-acylsynthetases and tRNAs. This review focuses on our current knowledge of protein synthesis in higher plants.

Amino Acid Sequence↗
PubMed1996

A statewide survey of hazards in child care centers.

OBJECTIVES: The purpose of this study was to determine adherence to selected recommended safety standards in North Carolina child care centers. METHODS: A self administered questionnaire eliciting information about safety practices in child care was mailed to a randomly selected sample of 409 North Carolina child care centers. RESULTS: One hundred and ninety five usable questionnaires were returned from child care centers in 75 counties. Results indicated that all of the standards included in the state's child regulations were being adhered to by at least 80% of the centers. However, adherence to recommended standards not included in the state's regulations was quite variable, with one standard implemented by less than 5% of the centers. The lowest rates of adherence were found for standards specifying that resilient surface material be used under playground equipment (4%) and that certain foods that may present a choking hazard to small children not be served (27%). CONCLUSIONS: Many hazards not addressed in North Carolina child care regulations are present in child care centers. Some safety standards are not adhered to due to lack of knowledge or limited resources. Inclusion of national standards in state child care regulations appears to reduce, but not eliminate, the likelihood of hazards being reported. Further research should include on-site inspections and attention to safety in family child care.

Child↗
PubMed1995

Interaction of wheat germ protein synthesis initiation factor eIF-(iso)4F and its subunits p28 and p86 with m7GTP and mRNA analogues.

The binding of p28, p86, and native wheat germ eIF-(iso)4F with m7GTP and oligonucleotides was measured and compared. The purified subunits (p28, 28 kDa and p86, 86 kDa) of wheat germ protein synthesis initiation factor eIF-(iso)4F have been obtained from Escherichia coli expression of the cloned DNA (van Heerden, A., and Browning, K. S. (1994) J. Biol. Chem. 269, 17454-17457). The binding of the 5'-terminal cap analogue m7GTP to the small subunit (p28) of eIF-(iso)4F as a function of pH, temperature, and ionic strength is described. The mode of binding of p28 to cap analogues is very similar to the intact protein. Assuming that all tryptophan residues contribute to p28 and eIF-(iso)4F fluorescence, iodide quenching shows that all 9 tryptophan residues in p28 are solvent-accessible, while only 6 out of 16 tryptophan residues are solvent-accessible on the intact eIF-(iso)4F. The fluorescence stopped-flow studies of eIF-(iso)4F and p28 with cap show a concentration-independent conformational change. The rate of this conformational change was approximately 10-fold faster for the isolated p28 compared with the native eIF-(iso)4F. From these studies it appears that cap recognition resides in the p28 subunit. However, p86 enhances the interaction with capped oligonucleotides and probably is involved in protein-protein interactions as well. Both subunits are required for helicase activity.

Base Sequence↗
PubMed1995

mRNA binding properties of wheat germ protein synthesis initiation factor 2.

The binding of protein synthesis initiation factor (eIF-) 2 to mRNA was measured by retention of the mRNA.eIF-2 complexes on nitrocellulose filters. The binding of eIF-2 to mRNA was inhibited by GDP and GTP; the inhibition by GTP was enhanced by the presence of Met-tRNA(i). In addition, the formation of eIF-2.GDP binary complex and eIF-2.GTP.Met-tRNA(i) ternary complex was inhibited by mRNA. These data indicate that mRNA binds to a site on eIF-2 that is the same or overlaps the site to which guanine nucleotides bind eIF-2. This finding strongly suggests that inhibition of ternary complex formation by mRNA may be the result of competition between mRNA and GTP and not competition between mRNA and Met-tRNA(i).

DNA↗
PubMed1995

Function of the p86 subunit of eukaryotic initiation factor (iso)4F as a microtubule-associated protein in plant cells.

The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of a p28 subunit that binds the 7-methylguanine cap of mRNA and a p86 subunit having unknown function. The p86 subunit was found to have limited sequence similarity to a kinesin-like protein encoded by the katA gene of Arabidopsis thaliana. Native wheat germ eIF-(iso)4F and bacterially expressed p86 subunit and p86-p28 complex bound to taxol-stabilized maize microtubules (MTs) in vitro. Binding saturation occurred at 1 mol of p86 per 5-6 mol of polymerized tubulin dimer, demonstrating a substoichiometric interaction of p86 with MTs. No evidence was found for a direct interaction of the p28 subunit with MTs. Unlike kinesin, cosedimentation of eIF-(iso)4F with MTs was neither reduced by MgATP nor enhanced by adenosine 5'-[gamma-imido]triphosphate. Both p86 subunit and p86-p28 complex induced the bundling of MTs in vitro. The p86 subunit was immunolocalized to the cytosol in root maize cells and existed in three forms: fine particles, coarse particles, and linear patches. Many coarse particles and linear patches were colocalized or closely associated with cortical MT bundles in interphase cells. The results indicate that the p86 subunit of eIF-(iso)4F is a MT-associated protein that may simultaneously link the translational machinery to the cytoskeleton and regulate MT disposition in plant cells.

Arabidopsis Proteins↗
PubMed1995

Determination of the amounts of protein synthesis initiation factors required for translation of rabbit alpha-globin and beta-globin mRNAs.

alpha-Globin mRNA was translated at 60-80% of the rate of beta-globin mRNA in a rabbit reticulocyte lysate, a wheat germ S30 and in a wheat germ partially purified system. The concentrations of the initiation factors (eIF-) required to obtain half-maximal rate of translation (C0.5) of these mRNAs were determined in the partially purified system from wheat germ. The C0.5s of eIF-3, eIF-2 and eIF-4F were found to be about the same. The C0.5s of eIF-4C and eIF-4A for alpha-globin mRNA were only slightly higher (1.5-fold) than those of beta-globin mRNA. The C0.5 for eIF-4B was 2.5-fold higher for alpha-globin mRNA. Addition of saturating amounts of these initiation factors to either the S-30 system or the partially purified system did not increase the rate of translation of alpha-globin mRNA. These results indicate that the difference in the rate of translation of alpha- and beta-globin mRNAs is not due solely to the amounts of factors required.

Animals↗
PubMed1994

Expression in Escherichia coli of the two subunits of the isozyme form of wheat germ protein synthesis initiation factor 4F. Purification of the subunits and formation of an enzymatically active complex.

The subunits (p28 and p86) of the isoenzyme form of eukaryotic initiation factor 4F (eIF-(iso)4F) from wheat were expressed separately in Escherichia coli. The subunits were purified by affinity chromatography (p28) and ion-exchange chromatography (p86). The purified subunits alone did not support polypeptide synthesis in an eIF-(iso)4F and eIF-4F-deficient translation system from wheat germ. However, when the two subunits were mixed together, activity equal to that of the native form of eIF-(iso)4F was obtained. These results show that subunits expressed separately are able to associate and form an enzymatically active complex.

Base Sequence↗
PubMed1993

Characterization of wheat germ protein synthesis initiation factor eIF-4C and comparison of eIF-4C from wheat germ and rabbit reticulocytes.

Eukaryotic protein synthesis initiation factor (eIF)-4C was purified from wheat germ and the molecular weight was calculated to be approximately 19,000 by SDS-polyacrylamide gel electrophoresis. A similar molecular weight was determined by gel filtration chromatography indicating that wheat germ eIF-4C is functional as a single polypeptide chain. An efficient in vitro translation system dependent upon the addition of eIF-4C was developed. This system was used to determine the concentrations of eIF-4C required for the half-maximal rate of translation of satellite tobacco necrosis virus RNA, alfalfa mosaic virus RNA 4, and barley alpha-amylase mRNA. No significant differences in the concentrations of eIF-4C required for the translation of these mRNAs were observed, although differences were noted for eIF-4A and eIF-4F. This finding suggests that eIF-4C is not involved in the binding of mRNA to 40 S ribosomal subunits. In heterologous assays, rabbit reticulocyte eIF-4C was as active as wheat germ eIF-4C in the wheat germ eIF-4C-dependent system. In addition, wheat germ eIF-4C substituted for rabbit reticulocyte eIF-4C in in vitro assay systems from rabbit reticulocytes. These results indicate that eIF-4C from wheat and rabbit contain conserved functional domains.

Animals↗
PubMed1993

Sequence of a cDNA encoding wheat eukaryotic protein synthesis initiation factor 4A.

A cDNA encoding wheat translation initiation factor 4A (eIF-4A) was isolated from a wheat cDNA library and sequenced. The deduced wheat eIF-4A amino acid sequence from the cDNA is compared to eIF-4A from Arabidopsis thaliana, tobacco, mouse and Saccharomyces cerevisiae. Putative RNA helicase motifs, and putative ATP-binding and hydrolysis sites are identified.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and expression of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase and sequence similarity with protein phosphatase 2C.

After many unsuccessful attempts to detect cDNA encoding the catalytic subunit of bovine pyruvate dehydrogenase phosphatase (PDPc) in bovine cDNA libraries, an approach based on the polymerase chain reaction (PCR) was undertaken. Overlapping DNA fragments were generated by PCR from bovine genomic DNA and from cDNA synthesized from total RNA with synthetic oligonucleotide primers on the basis of experimentally determined amino acid sequences. The DNA fragments were subcloned and sequenced. The complete cDNA is 1900 base pairs in length and contains an open reading frame of 1614 nucleotides encoding a putative presequence of 71 amino acid residues and a mature protein of 467 residues with a calculated M(r) of 52,625. Hybridization analysis showed a single mRNA transcript of about 2.0 kilobases. Comparison of the deduced amino acid sequences of the mitochondrial PDPc and the rat cytosolic protein phosphatase 2C indicates that these protein serine/threonine phosphatases evolved from a common ancestor. The mature form of PDPc was coexpressed in Escherichia coli with the chaperonin proteins groEL and groES. The recombinant protein (rPDPc) was purified to near homogeneity. Its activity toward the bovine 32P-labeled pyruvate dehydrogenase complex was Mg(2+)-dependent and Ca(2+)-stimulated and comparable to that of native bovine PDP. An active, truncated form of rPDPc, with M(r) approximately 45,000, was produced in variable amounts during growth of cells and/or during the purification procedure.

Amino Acid Sequence↗
PubMed1993

The 5' and 3' untranslated regions of satellite tobacco necrosis virus RNA affect translational efficiency and dependence on a 5' cap structure.

Satellite tobacco necrosis virus RNA (STNV RNA) is a naturally uncapped viral RNA that contains 1239 nucleotides: 29 in the 5' untranslated region (UTR), 591 in the coding region and 619 in the 3' UTR. Mutations were made in the 5' and 3' UTRs, and the effects of these mutations on translational efficiency and cap independence were measured in an in vitro translation system from wheat germ. Removal of the first 12 nucleotides or 10 changes in the nucleotide sequence of the 5' UTR reduced translational efficiency approximately 3-fold; capping of these 5' mutant mRNAs restored their translational efficiencies. Truncation of the 3' UTR to nucleotide 627 or 700, or deletion of nucleotides 627-737, reduced translational efficiency more than 20-fold; capping of these 3' mutant mRNAs restored their translational efficiencies. These modifications in the 3' UTR increased the concentration of initiation factor 4F required for translation. Chimeric mRNAs were constructed which contained the coding region of rabbit alpha-globin mRNA and either the 5' UTR, 3' UTR, or both the 5' and 3' UTRs of STNV RNA. Both the 5' and 3' UTRs of STNV RNA were necessary to obtain cap-independent translation. These findings indicate that interaction between 5' UTR and the region between nucleotides 627 and 737 in the 3' UTR are required for cap-independent translation.

Animals↗
PubMed1993

Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase.

The association of host proteins with viral RNA replication proteins has been reported for a number of (+)-strand RNA viruses. However, little is known about the identity or function of these host proteins in viral replication. In this paper we report the characterization of a host protein associated with the RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley. A host protein was specifically and proportionally enriched with BMV RdRp activity through several purification steps. This RdRp-associated host protein reacted with an antiserum prepared against wheat germ eukaryotic translation initiation factor 3 (eIF-3). The RdRp-associated host protein, the p41 subunit of wheat germ eIF-3, and an antigenically related protein from rabbit reticulocyte lysates were all found to bind with high affinity and specificity to BMV-encoded protein 2a, which is involved in viral RNA replication. Moreover, addition of wheat germ eIF-3 or the p41 subunit from wheat germ to BMV RdRp gave a specific and reproducible 3-fold stimulation of (-)-strand RNA synthesis in vivo. These results suggest that the barley analog of eIF-3 subunit p41, or a closely related protein, associates with BMV RdRp in vivo and is involved in BMV RNA replication. This observation and the established role of translation factors in bacteriophage Q beta RdRp suggest that association with translation factors may be a general feature of RNA replication by (+)-strand RNA viruses.

Animals↗
PubMed1992

Isolation and sequence of the cDNAs encoding the subunits of the isozyme form of wheat protein synthesis initiation factor 4F.

The nucleotide sequences of the cDNAs for the two subunits, p82 and p28, of the isozyme form of wheat germ eukaryotic initiation factor 4F (eIF-(iso)4F) were determined. The cDNA for the p82 subunit encodes a polypeptide of 86,514 Da. The deduced amino acid sequence of p82 contains possible motifs for ATP binding, metal binding, and phosphorylation. The cDNA sequence for the small subunit, p28, which is a m7G cap-binding protein, encodes a polypeptide of 23,524 Da. The deduced amino acid sequence of p28 is similar (approximately 38%) to cap-binding proteins from yeast and mammals. The p28 of wheat eIF-(iso)4F does not contain a serine or threonine in the vicinity of the serine (Ser53) of mammalian cap-binding protein which is phosphorylated and shown to affect activity in mammalian cells.

Amino Acid Sequence↗