Biomedical subjects

J W Bodley

Publications and source records attributed to J W Bodley.

At least 19 recordsLinked to original sources

PubMed1996

Expression, purification, and characterization of the G domain of Saccharomyces cerevisiae elongation factor 2.

Protein synthesis elongation factor 2 (EF-2), a member of the G protein superfamily, catalyzes the movement of the ribosome along mRNA in a reaction driven by the hydrolysis of GTP. In the present study, we have expressed, purified, and characterized the G domain of Saccharomyces cerevisiae EF-2 to define its functional properties. A peptide of 185 amino acids was found to be sufficient to bind guanine nucleotides when expressed in Escherichia coli either as a fusion with the maltose binding protein or as a truncation. The fusion was expressed as a soluble, active protein, while the truncation was expressed as an insoluble protein that required renaturation in order to function. Similar to native EF-2, both expression products bound GDP, but they did so with significantly lower affinity (Kd ca. 200 microM for both the fusion and the truncation vs ca. 0.5 microM for native EF-2). However, in contrast to native EF-2 which requires the ribosome for GTP hydrolysis, the isolated G domain hydrolyzed GTP (Km ca. 40 microM; turnover rate ca. 0.5 min-1) in the absence of the ribosome. We conclude that the G domain residues necessary for GTP binding and hydrolysis are located in the first 185 amino acids of S. cerevisiae EF-2.

Cloning, Molecular↗

PubMed1994

Differential transcription of the two Saccharomyces cerevisiae genes encoding elongation factor 2.

The single polypeptide chain of elongation factor 2 (EF-2) is encoded by two Saccharomyces cerevisiae genes (EFT1 and EFT2) with unique flanking sequences. One gene is necessary and either is sufficient for cell viability. In the present work, we have analyzed the transcription of EFT1 and EFT2. Although both genes harbor multiple transcription start points, EFT1 initiates primarily at residue C at -39 and EFT2 at residue C at -37. Several candidate TATA boxes were identified in each gene. Deletion analysis employing lacZ promoter fusions demonstrated that the promoter for EFT2 is located within a 79-bp region beginning 335 nucleotides (nt) upstream from the start ATG codon. This region contains two overlapping sequences with homology to the consensus binding site for the yeast transcription factor, Rap1p/Grf1p/Tuf. In contrast, the sequences essential for the transcription of EFT1 were localized to the region between the start ATG and the stop codon of the VPS17 gene that terminates 267 nt upstream on the same strand. Analysis of promoter strengths using lacZ fusions indicated that the promoter for EFT2 is approx. 2.5-fold more active than that of EFT1. Analysis of the steady-state levels of mRNAs revealed that EFT2 contributes approx. 70% of the total EF-2 mRNA while the remaining 30% is produced by EFT1. We conclude that the difference in expression of EFT1 and EFT2 is due to the differential transcription of their promoters.

Base Sequence↗

PubMed1994

Identification of bacteriophage phi 29 prohead RNA domains necessary for in vitro DNA-gp3 packaging.

Functional domains of the bacteriophage phi 29 prohead RNA (pRNA) that are essential for in vitro packaging of DNA-gp3 into the prohead were mapped using pRNA mutants. Oligonucleotide-directed mutant pRNAs were produced that contained deletions and sequence alterations but were predicted to retain the overall secondary structure of wild-type pRNA. Mutant pRNAs were compared to wild-type pRNA for prohead binding in a competition assay and for DNA packaging in the defined in vitro system. The prohead binding site was previously localized to residues 22-84 on the 120-residue domain I of pRNA by ribonuclease footprinting (Reid, R. J. D., Bodley, J. W., and Anderson, D. (1994) J. Biol. Chem. 269, 5157-5162). Mutations of pRNA within the prohead binding site resulted in substantial loss of prohead binding capacity, while mutations outside of the footprint had moderate effects on prohead binding. DNA-gp3 packaging activity was correlated with pRNA binding activity for mutations within the footprint. This mutational analysis showed that both sequence and secondary structure of residues 40-91 of pRNA were crucial for prohead binding and that elements of the A helix formed from residues 1-28 and 117-92 were needed for DNA packaging functions other than prohead binding.

Bacillus Phages↗

PubMed1994

Characterization of the prohead-pRNA interaction of bacteriophage phi 29.

The small prohead RNA (pRNA) of the Bacillus subtilis bacteriophage phi 29 is essential for ATP-dependent packaging of viral DNA. The 174-, 124-, and 120-residue forms of pRNA produced in vitro using T7 RNA polymerase were equivalent in prohead binding and DNA packaging activity to pRNAs produced in phi 29-infected cells. pRNA binding to proheads, characterized by the use of Northern hybridization and filter binding assays, was specific, rapid, and irreversible in the presence of 10 mM Mg2+. Proheads produced in phage-infected cells carried 5.8 +/- 2.7 copies of pRNA, and proheads assembled in Escherichia coli in the absence of pRNA bound 6.0 +/- 3.5 copies of pRNA. Footprints of proheads on pRNA generated with the ribonucleases A, T1, and V1 showed that nucleotides 22-84, 5' to 3', were protected from ribonuclease attack. Enhanced cleavage at nucleotides 37-40 with ribonuclease V1 suggested a conformational change of pRNA upon prohead binding.

Bacillus Phages↗

PubMed1993

Saccharomyces cerevisiae elongation factor 2. Mutagenesis of the histidine precursor of diphthamide yields a functional protein that is resistant to diphtheria toxin.

Protein synthesis elongation factor 2 (EF-2) is the target of the ADP-ribosylating activity of diphtheria toxin which is responsible for cell killing. Diphthamide, an unique post-translationally modified histidine residue, is both required for and the site of this ADP-ribosylation. Although present in the EF-2 of all eukaryotes and archaebacteria, the function of diphthamide is unknown. Here we describe the site-specific mutagenesis of the histidine precursor of diphthamide, histidine 699, in yeast EF-2. Plasmid-borne EFT was randomly mutagenized at the histidine 699 codon, and the technique of plasmid shuffling was utilized to select strains that were maintained by the mutant EFT. These mutants were screened for diphtheria toxin resistance. Sequence analysis of the EFT in 49 toxin-resistant isolates showed that histidine 699 had been replaced by 1 of 4 amino acids: asparagine, glutamine, leucine, or methionine. All 11 of the possible codons corresponding to these 4 amino acids were found. The growth rates of cells sustained by the mutant forms of EF-2 were slightly slower than those of isogenic wild-type cells. We conclude that despite its strict conservation and universal post-translational modification, the histidine precursor of diphthamide is not essential to the function of yeast EF-2 in protein synthesis.

Base Sequence↗

PubMed1992

Saccharomyces cerevisiae elongation factor 2. Genetic cloning, characterization of expression, and G-domain modeling.

The elongation factor 2 (EF-2) genes of the yeast Saccharomyces cerevisiae have been cloned and characterized with the ultimate goal of gaining a better understanding of the mechanism and control of protein synthesis. Two genes (EFT1 and EFT2) were isolated by screening a bacteriophage lambda yeast genomic DNA library with an oligonucleotide probe complementary to the domain of EF-2 that contains diphthamide, the unique posttranslationally modified histidine that is specifically ADP-ribosylated by diphtheria toxin. Although EFT1 and EFT2 are located on separate chromosomes, the DNA sequences of the two genes differ at only four positions out of 2526 base pairs, and the predicted protein sequences are identical. Genetic deletion of each gene revealed that at least one functional copy of either EFT gene is required for cell viability. Messenger RNA levels of yeast EF-2 parallel cellular growth and peak in mid-log phase cultures. The EF-2 protein sequence is strikingly conserved through evolution. Yeast EF-2 is 66% identical to, and shares over 85% homology with, human EF-2. In addition, yeast and mammalian EF-2 share identical sequences at two critical functional sites: (i) the domain containing the histidine residue that is modified to diphthamide and (ii) the threonine residue that is specifically phosphorylated in vivo in mammalian cells by calmodulin-dependent protein kinase III, also known as EF-2 kinase. Furthermore, yeast EF-2 also contains the Glu-X-X-Arg-X-Ile-Thr-Ile "effector" sequence motif that is conserved among all known elongation factors, and its GTP-binding domain exhibits strong homology to the G-domain of Escherichia coli elongation factor Tu (EF-Tu) and other G-protein family members. Based upon these observations, we have modeled the G-domain of the deduced EF-2 protein sequence to the solved crystallographic structure for EF-Tu.

Amino Acid Sequence↗

PubMed1992

A screening procedure for the intracellular expression of native proteins by Saccharomyces cerevisiae: discrimination of diphtheria toxin-resistant mutants.

A general method is described for screening Saccharomyces cerevisiae colonies for the intracellular expression of native proteins. Colonies are replicated onto nitrocellulose membranes and yeast cell walls are removed enzymatically. The resulting spheroplasts are rapidly lysed by placing chromatography paper soaked in hypotonic buffer on the membranes. Intracellular proteins released by spheroplast lysis are bound in situ to the nitrocellulose under non-denaturing conditions and potentially can be examined using enzymatic or immunologic methods. For example, in the present study colonies were screened for the presence of elongation factor 2 (EF-2) that can be [32P]ADP-ribosylated by diphtheria toxin and [32P]NAD+. Recognition by the toxin requires the presence in EF-2 of the unique post-translationally modified histidine derivative, diphthamide. The procedure described here reliably discriminates between wild-type yeast colonies and mutant colonies that do not synthesize diphthamide. In addition to facilitating the study of diphthamide biosynthesis in yeast, the more general application of this procedure will enable the screening of colonies with assays that require native proteins.

Collodion↗

PubMed1992

Protein toxin inhibitors of protein synthesis.

Two classes of extremely toxic proteins kill eukaryotic cells by covalently modifying unique structural features of components that are essential for protein synthesis. Intoxication by these proteins results from the entry of a catalytic fragment into the cytoplasm. One class is typified by diphtheria toxin and Pseudomonas exotoxin A. The catalytic component of these toxins ADP-ribosylates and inactivates elongation factor 2 which is an essential participant in protein synthesis. This modification occurs at a unique post-translational histidine derivative, diphthamide, that is present in the ribosomal binding site of the elongation factor. The two toxins differ in their molecular organization but appear to possess identical reaction mechanisms and very similar active sites. The other class contains two types of toxins typified, respectively, by alpha-sarcin, a member of a family of fungal toxins, and ricin, a member of a group of closely related plant proteins collectively termed ribosome-inactivating proteins. The catalytic components of the two types of toxins in this second class inactivate the large ribosomal subunit through two different hydrolytic alterations of 23-28S RNA. alpha-Sarcin and its congeners act as a specific endonuclease whereas ricin and its congeners act as a specific N-glycosidase. These hydrolytic cleavages occur at a pair of adjacent nucleotides within a highly conserved sequence near the 3' terminus of 23-28S RNA. The covalent integrity of this region of RNA is essential to elongation factor-dependent ribosomal functions and is located within the ribosomal binding domain of these factors. Both of these classes of toxins are being employed as 'magic bullets' to eliminate pathological cells. By combining the catalytic component of these toxins with various cell targeting components, useful and specific anticancer and immunomodulatory agents have been created.

Histidine↗

PubMed1991

Saccharomyces cerevisiae elongation factor 2 is phosphorylated by an endogenous kinase.

Mammalian cells contain a Ca2+/calmodulin-dependent protein kinase that specifically phosphorylates and inactivates elongation factor 2 (EF-2) in response to hormones and other agents which increase intracellular Ca2+ concentrations. Therefore, it has been proposed that the rate of translation in mammals is regulated by EF-2 phosphorylation. In the present study, EF-2 purified from the yeast Saccharomyces cerevisiae is shown to be a substrate for the mammalian EF-2 kinase. Furthermore, evidence was obtained using two-dimensional gel electrophoresis and peptide mapping which suggests that yeast EF-2 is a substrate for an endogenous kinase which phosphorylates the same site as the mammalian EF-2 kinase. Based on these findings, we propose that in yeast as in higher eukaryotes, the protein synthesis elongation cycle is regulated by phosphorylation of EF-2.

Animals↗

PubMed1991

Alpha-sarcin cleavage of ribosomal RNA is inhibited by the binding of elongation factor G or thiostrepton to the ribosome.

The translocation reaction catalyzed by elongation factor G (EF-G) is inhibited either by alpha-sarcin cleavage of 23S rRNA or by the binding of thiostrepton to the E. coli ribosome. Here we show that the transitory binding of EF-G and GDP to the ribosome inhibited the rate of alpha-sarcin cleavage and that stabilization of this binding with fusidic acid completely prevented alpha-sarcin cleavage. A similar pattern of inhibition was seen upon the binding of elongation factor 2 to the S. cerevisiae ribosome. The irreversible binding of the antibiotic thiostrepton to the E. coli ribosome, on the other hand, decreased the rate of cleavage by alpha-sarcin approximately 2-fold. These results suggest that the alpha-sarcin site is located within the ribosomal domain for EF-G binding and that the conformation of this site is affected by the binding of thiostrepton.

Aspergillus↗

PubMed1990

Phylogenetic analysis and secondary structure of the Bacillus subtilis bacteriophage RNA required for DNA packaging.

An unusual RNA molecule encoded by the Bacillus subtilis bacteriophage phi 29 is a structural component of the viral prohead and is required for the ATP-dependent packaging of DNA. Here we report a model of secondary structure for this prohead RNA developed from a phylogenetic analysis of the primary sequences of prohead RNAs of related phages. Twenty-nine phages related to phi 29 were found to produce prohead RNAs. These RNAs were analyzed by their ability to replace phi 29 RNA in in vitro phage assembly, by Northern blot hybridization with a probe complementary to phi 29 RNA, and by partial and complete sequence analyses. These analyses revealed four quite different sequences ranging in length from 161 to 174 residues. The secondary structure deduced from these sequences, in agreement with earlier observations, indicated that prohead RNA is organized into two domains. The larger 5'-domain (Domain I) is composed of 113-117 residues and contains four helices. Three of these helices appear to be organized into a central stem that is interrupted by two unpaired loops and the fourth helix and loop. The smaller 3'-domain (Domain II) is composed of 40-44 residues and consists of two helices. Domains I and II are separated by 8-13 unpaired residues. Nuclease cleavage occurs readily in this single-stranded joining region, and this cleavage allows the subsequent separation of the two RNA domains. The separated Domain I is fully active in DNA packaging in vitro. The functional significance and biological role of Domain II are unknown. The phylogenetic secondary structure model provides a basis for further analysis of the role of this RNA in bacteriophage morphogenesis.

Bacillus subtilis↗

PubMed1989

Prohead RNA of bacteriophage phi 29: size, stoichiometry and biological activity.

We previously demonstrated (Guo et al., 1987. Nucl. Acids Res. 15, 7081-7090) that purified proheads of bacteriophage phi 29 contain an RNA of 120 bases which is essential for DNA packaging. Here we report that this RNA exists primarily as a polymer of ca. 174 residues in phage-infected cells and that ca. 54 bases are cleaved from its 3'-terminus by adventitious nucleases during the purification of proheads. The long and short forms of the RNA had similar activity in in vitro DNA packaging and phage assembly. We report the sequence of the long form of the RNA and show that similar long and short forms can be isolated from the proheads of the phi 29 relatives phi 21, phi 15 and SF5. The concentration dependence in the reconstitution of RNA-free proheads suggests that one copy of the RNA is sufficient to restore DNA packaging activity to RNA-free proheads. However, quantitative measurements indicate that 5 to 6 copies of the RNA are present on proheads isolated from phage-infected cells.

Bacillus subtilis↗

PubMed1988

Biosynthesis of diphthamide in Saccharomyces cerevisiae. Partial purification and characterization of a specific S-adenosylmethionine:elongation factor 2 methyltransferase.

The inactivation of elongation factor 2 (EF-2) by diphtheria toxin requires the presence of a post-translationally modified histidine residue in EF-2. This residue, diphthamide, has the structure 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine. The present work was undertaken to study the pathway of diphthamide biosynthesis using diphtheria toxin-resistant yeast mutants (Chen. J.-Y., Bodley, J. W., and Livingston, D. M. (1985) Mol. Cell. Biol. 5, 3357-3360) which are defective in diphthamide formation. We demonstrate here that one of these mutants (dph5) contains a toxin-resistant form of EF-2 which can be converted in vitro to a toxin-sensitive form through the action of an enzyme present in other yeast strains. Both this toxin-resistant EF-2 and its modifying enzyme have been partially purified and evidence is presented that the modifying enzyme is a specific S-adenosylmethionine:EF-2 methyltransferase. In vitro complementation to diphtheria toxin sensitivity required S-adenosylmethionine, and when partially purified components were incubated with [methyl-3H]S-adenosylmethionine, label was incorporated specifically into EF-2. Hydrolysis of labeled EF-2 yielded diphthine (the unamidated form of diphthamide) and a single chromatographically separable labeling intermediate. We conclude that the S-adenosylmethionine:EF-2 methyltransferase adds at least the last two of the three methyl groups present in diphthine and that this modification is sufficient to create diphtheria toxin sensitivity. Evidence is also presented for the existence of an ATP-dependent amidating enzyme which catalyzes the final step in the biosynthesis of diphthamide in EF-2.

Adenosine Diphosphate Ribose↗

PubMed1988

Alpha-sarcin cleaves ribosomal RNA at the alpha-sarcin site in the absence of ribosomal proteins.

alpha-Sarcin is capable of specifically cleaving the single phosphodiester bond in the "alpha-sarcin site" of both Escherichia coli and Saccharomyces cerevisiae large rRNAs in the absence of ribosomal proteins. With both sources of rRNA, the rate of cleavage was comparable with and without ribosomal proteins but more complete cleavage was observed in the absence of ribosomal proteins. These observations contrast with earlier findings and indicate that ribosomal proteins are not essential to the unique specificity of the cleavage of rRNA by alpha-sarcin.

Endoribonucleases↗

PubMed1988

The ribosomes of Aspergillus giganteus are sensitive to the cytotoxic action of alpha-sarcin.

Ribosomes in lysates prepared from the mycelia of Aspergillus giganteus MDH 18894, which are actively secreting alpha-sarcin, do not contain the alpha-sarcin lesion. However, the addition of exogenous alpha-sarcin to these same lysates results in cleavage of the 26 S rRNA of the 60 S ribosomal subunit, characteristic of the cytotoxic action of alpha-sarcin. We conclude that A. giganteus ribosomes are not inherently resistant to the action of alpha-sarcin but are protected in vivo by producing alpha-sarcin in an inactive form and/or by the efficient cotranslational secretion of the toxin.

Aspergillus↗

PubMed1988

Modification of ribosomal RNA by ribosome-inactivating proteins from plants.

We have surveyed 14 different toxic and nontoxic ribosome-inactivating proteins from plants for the ability to act on the RNA of the eucaryotic 60 S ribosomal subunit. All of these proteins act to introduce a specific modification into 26-28 S RNA which renders the RNA sensitive to cleavage by aniline. Sequence analysis of the 5'-termini of the fragments produced by ricin and saporin following aniline cleavage indicate that both proteins possess identical specificity. Our observations support the conclusion of Endo and Tsurugi (J. Biol. Chem. 262, 8128-8130, 1987) that ricin is a specific N-glycosidase and we have located the site of this cleavage by direct sequence analysis. Our results further suggest that all plant ribosome-inactivating proteins function as specific N-glycosidases with the same specificity.

Animals↗

PubMed1988

Expression of diphtheria toxin fragment A and hormone-toxin fusion proteins in toxin-resistant yeast mutants.

Mutants of the eukaryote Saccharomyces cerevisiae, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins. The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL1 promoter element in a plasmid. Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A. In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GAL1 promoter was induced. Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or alpha-melanocyte-stimulating hormone along with the lipid-associating domains of fragment B and the leader peptide of the Kluyveromyces lactis killer toxin. Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.

Amino Acid Sequence↗

PubMed1987

Characterization of the small RNA of the bacteriophage phi 29 DNA packaging machine.

The prohead connector of the bacteriophage luminal diameter 29 DNA packaging machine was reconstructed with the small RNA that regulates DNA packaging in vitro. The complete sequence of the 120 nucleotide RNA proved its origination from the promoter PE1(A1) of the left early region of phi 29 DNA, the end packaged first during assembly. The prohead RNA was clearly distinct from eubacterial 5S rRNA in sequence and composition.

Bacillus subtilis↗