A key component in determining the functional role of any protein is the elucidation of its bindi... more A key component in determining the functional role of any protein is the elucidation of its binding partners using protein-protein interaction (PPI) data. Here we examine the use of tandem affinity purification (TAP) tagging to study RNA/DNA helicase PPIs in Escherichia coli. The tag, which consists of a calmodulin-binding region, a TEV protease recognition sequence, and an IgG-binding domain, is introduced into E. coli using a lambdared recombination system. This method prevents the overproduction of the target protein, which could generate false interactions. The interacting proteins are then affinity purified using double affinity purification steps and are separated by SDS-PAGE followed by mass spectrometry identification. Each protein identified would represent a physical interaction in the cell. These interactions may potentially be mediated by an RNA/DNA template, for which the helicase would likely be needed to disrupt the secondary structures.
The in vitro reversal of conditionally lethal mutations has greatly aided the study of translatio... more The in vitro reversal of conditionally lethal mutations has greatly aided the study of translation. N4316 is a mutant of Escherichia coli that has a temperature-sensitive defect in a protein called the rescue protein. Without the rescue protein, translation in vivo and in vitro is drastically reduced and frameshift errors, as well as increased read-through of nonsense codons, occurs. Using reversal of temperature-sensitivity as an assay, the rescue protein was purified from a ribosomal eluate of the parental (D10) strain. Composite polyacrylamide/agarose gel electrophoresis and sedimentation on sucrose density gradients were employed to examine the distribution of 70s ribosomes and ribosomal subunits in the mutant (N4316) and the parental (D10) extracts at restrictive (43°C) and non-restrictive (35 "C) temperatures. Fewer polysomes and a larger proportion of 70s ribosomes relative to subunits were observed at 43°C with N4316, but not with D10 extracts. Addition of the rescue protein had no effect at 35°C with either strain, but restored the polysome pattern of N4316 at 43°C. The purified rescue protein labelled by methylation retained activity and bound preferentially to 30s subunits. Rescue bound to 30s particles prevented the action of IF-3 fostering formation of 70s ribosomes. Thus the rescue protein enables formation of 70s ribosomes from 30s and 50s subunits. 70s ribosomes which contain the rescue protein are active in translation and resist dissociation induced by high centrifugal fields. We propose that the rescue protein alters the conformation of 70s ribosomes resulting in a tighter association of subunits which, in turn, fosters both higher rates and increased accuracy of translation. Conditionally lethal mutants have been extremely useful in studies of the mechanisms underlying translation [ 1-31. One such mutant is strain N4316 which is derived from the Escherichia coli thymidine auxotroph D10 (met-, thy-, RNase I-). N4316 was isolated by a procedure designed to optimize the selection for temperature-sensitive ribosomal mutants [4, 51. At 43"C, translation in vivo is impaired and N4316 cells die over a period of a few hours [4]. At 36"C, N4316 suppresses termination codons UGA and UAA, and occasionally UAG, as well as certain missense codons [4, 51. The temperature-sensitive growth and temperature-dependent suppression are believed to be due to lesions in two separate genes (sts and sut, respectively) which map at different chromosomal loci and can be separated by transduction and cloning [6-81. At restrictive temperatures [4], crude extracts of
Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translatio... more Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translation elongation in some lower fungi. Here we report the first identification of a ribosome bound ATPase, RbbA, in E. coli cells. RbbA accounts for most of the ATPase activity associated with 70S ribosomes and 30S ribosomal subunits. Both native and recombinant RbbA were purified and shown to possess ribosome-dependent ATPase activities and to stimulate polyphenylalanine synthesis in vitro. Biochemically, RbbA is similar to the fungi-specific translation elongation factor 3 (EF-3) and cross-reacts with antibody raised against EF-3. The gene encoding RbbA is identified as ORF yhih and the predicted RbbA amino acid sequence is 40% similar to that of the C-terminal half of EF-3. The discovery of a ribosomal ATPase in a prokaryotic cell suggests a common, conserved function for these proteins in translation.
Biochemical and Biophysical Research Communications, 2005
The oxazolidinones are a new class of potent antibiotics that are active against a broad spectrum... more The oxazolidinones are a new class of potent antibiotics that are active against a broad spectrum of Gram-positive bacterial pathogens including those resistant to other antibiotics. These drugs specifically inhibit protein biosynthesis whereas DNA and RNA synthesis are not affected. Although biochemical and genetic studies indicate that oxazolidinones target the ribosomal peptidyltransferase center, other investigations suggest that they interact with different regions of ribosomes. Thus, the exact binding site and mechanism of action have remained elusive. Here, we study, by use of base-specific reagents, the effect of the oxazolidinones on the chemical protection footprinting patterns of the 23S rRNA. We report: (i) reproducible protection of G2607 and G2608 of 23S rRNA by a potent oxazolidinone on a ribosomeAEtRNAAEmRNA complex; (ii) no protections were observed on 70S ribosomes devoid of tRNA and mRNA; (iii) EF-G also weakly protected G2607 and G2608; (iv) mutations at G2608 conferred resistance to the oxazolidinones in Escherichia coli cells; and (v) G2607 and G2608 occur near the exit to the peptide tunnel on the 50S subunit. A mechanism for the pleiotropic action of the oxazolidinones is discussed.
Bacillus amyloliquefaciens strain AL 35, produced high yields of a cyclodextrin glycosyltransfera... more Bacillus amyloliquefaciens strain AL 35, produced high yields of a cyclodextrin glycosyltransferase (CGT'ase) when grown in a submerged culture. The stability of CGT'ase to high temperature and alkaline pH enabled processing for cyclodextrin production to be carried out at 60°C and pH 9.0. Crude culture filtrates containing the CGT'ase could convert gelatinized starch substrates to predominantly a-cyclodextrins (up to 95% of the total cyclodextrin yields).
The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine–threonine kinase, en... more The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine–threonine kinase, encompassing three catalytic (Tpk1–3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the tpk1 catalytic subunit reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ factor (nej1), co-function in the same pathway, and parallel to the NHEJ factor yku80. Chromatin immunoprecipitation and resection data suggest that tpk1 deletion influences repair protein recruitments and DNA resection. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair and nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKA...
2-Methylcitrate dehydratase (2-methylcitrate hydro-lyase) functioning at the methylcitric acid cy... more 2-Methylcitrate dehydratase (2-methylcitrate hydro-lyase) functioning at the methylcitric acid cycle of propionyl-CoA oxidation was purified from a cell-free extract of Yarrowia
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1987
l. fl-N-acetylglucosaminidase and one of the several chitinases were purified from the supernatan... more l. fl-N-acetylglucosaminidase and one of the several chitinases were purified from the supernatant fraction from homogenates of pupae of the red flour beetle, Tribolium castaneum Herbst, by ammonium sulfate fractionation, hydroxylapatite chromatography, anion exchange chromatography and gel filtration chromatography. 2. //-N-acetylglucosaminidase exhibited a dimeric structure with apparent subunit molecular weights of 7.3 x 104 and 6.4 x 104, whereas chitinase was a monomer with an apparent mol. wt of 7.7 x 104. 3. //-N-acetylglucosaminidase and chitinase were composed of approximately 1200 and 700 amino acid residues per molecule and exhibited isoelectric points of pH 4.9 and 4.7, respectively. 4. The kcat/K m value for fl-N-acetylglucosaminidase hydrolyzing flGIcNAc 2 was 2.22 x 104 M-I see-I while that for chitinase hydrolyzing glycol chitin was 6.4 rnl mg-l see-t. 5. The results demonstrate a chitinolytic enzyme system in 7". castaneum composed of an endo-splitting chitinase and an exo-splitting fl-N-acetylglucosaminidase. *Contribution No. 86-324-J. Department of Biochemistry, Kansas Agricultural Experiment Station, Manhattan, Kansas 66506. Cooperative investigation between ARS, USDA and the Kansas Agricultural Experiment Station. Mention of a proprietary product does not imply approval of this product by the USDA to the exclusion of other products that may also be suitable.
Disruption of eshA , which encodes a 52-kDa protein that is produced late during the growth of St... more Disruption of eshA , which encodes a 52-kDa protein that is produced late during the growth of Streptomyces coelicolor A3(2), resulted in elimination of actinorhodin production. In contrast, disruption of eshB , a close homologue of eshA , had no effect on antibiotic production. The eshA disruptant accumulated lower levels of ppGpp than the wild-type strain accumulated. The loss of actinorhodin production in the eshA disruptant was restored by expression of a truncated relA gene, which increased the ppGpp level to the level in the wild-type strain, indicating that the reduced ppGpp accumulation in the eshA mutant was solely responsible for the loss of antibiotic production. Antibiotic production was also restored in the eshA mutant by introducing mutations into rpoB (encoding the RNA polymerase β subunit) that bypassed the requirement for ppGpp, which is consistent with a role for EshA in modulating ppGpp levels. EshA contains a cyclic nucleotide-binding domain that is essential for...
Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of ... more Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus . Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent ( rel + ; wild type) and relaxed ( relA and relC ; mutant) strains of T. thermophilus . We found that in wild-type T. thermophilus , as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA Ser aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA -null mutant and partially blocked in a relC mutant harboring a muta...
We have previously isolated and described an Escherichia coli ribosome-bound ATPase, RbbA, that i... more We have previously isolated and described an Escherichia coli ribosome-bound ATPase, RbbA, that is required for protein synthesis in the presence of ATP, GTP and the elongation factors, EF-Tu and EF-G. The gene encoding RbbA, yhih, has been cloned and the deduced protein sequence harbors two ATP-motifs and one RNA-binding motif and is homologous to the fungal EF-3. Here, we describe the isolation and assay of a truncated form of the RbbA protein that is stable to overproduction and purification. Chemical protection results show that the truncated RbbA specifically protects nucleotide A937 on the 30S subunit of ribosomes, and the protected site occurs at the E-site where the tRNA is ejected upon A-site occupation. Other weakly protected bases in the region occur at or near the mRNA binding site. Using radiolabeled tRNAs, we study the stimulating effect of this truncated RbbA on the binding and release of different tRNAs bound to the (aminoacyl) A-, (peptidyl) P-and (exit) E-sites of 70S ribosomes. The combined data suggest plausible mechanisms for the function of RbbA in translation.
The oxazolidinones represent a new class of antimicrobial agents which are active against multidr... more The oxazolidinones represent a new class of antimicrobial agents which are active against multidrug-resistant staphylococci, streptococci, and enterococci. Previous studies have demonstrated that oxazolidinones inhibit bacterial translation in vitro at a step preceding elongation but after the charging of N -formylmethionine to the initiator tRNA molecule. The event that occurs between these two steps is termed initiation. Initiation of protein synthesis requires the simultaneous presence of N -formylmethionine-tRNA, the 30S ribosomal subunit, mRNA, GTP, and the initiation factors IF1, IF2, and IF3. An initiation complex assay measuring the binding of [ 3 H] N -formylmethionyl-tRNA to ribosomes in response to mRNA binding was used in order to investigate the mechanism of oxazolidinone action. Linezolid inhibited initiation complex formation with either the 30S or the 70S ribosomal subunits from Escherichia coli . In addition, complex formation with Staphylococcus aureus 70S tight-co...
G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with ke... more G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable re...
Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play cru... more Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provide...
Bacterial protein synthesis is an essential, conserved, and environmentally responsive process. Y... more Bacterial protein synthesis is an essential, conserved, and environmentally responsive process. Yet, many of its components and dependencies remain unidentified. To address this gap, we used quantitative synthetic genetic arrays to map functional relationships among >48,000 gene pairs in Escherichia coli under four culture conditions differing in temperature and nutrient availability. The resulting data provide global functional insights into the roles and associations of genes, pathways, and processes important for efficient translation, growth, and environmental adaptation. We predict and independently verify the requirement of unannotated genes for normal translation, including a previously unappreciated role of YhbY in 30S biogenesis. Dynamic changes in the patterns of genetic dependencies across the four growth conditions and data projections onto other species reveal overarching functional and evolutionary pressures impacting the translation system and bacterial fitness, un...
Highlights d Analyses of ubiquitous protein complexes identified new components in ASD d HDAC1/2 ... more Highlights d Analyses of ubiquitous protein complexes identified new components in ASD d HDAC1/2 positively regulates ASD orthologs in the mouse embryonic brain d IP/MS in neuronal cells identified protein complexes in ASD d A network bridges the gap between the idiopathic and syndromic forms of ASD
The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrit... more The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast ( Saccharomyces cerevisiae ) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identifie...
EF-P (eubacterial elongation factor P) is a highly conserved protein essential for protein synthe... more EF-P (eubacterial elongation factor P) is a highly conserved protein essential for protein synthesis. We report that EF-P protects 16S rRNA near the G526 streptomycin and the S12 and mRNA binding sites (30S T-site). EF-P also protects domain V of the 23S rRNA proximal to the A-site (50S T-site) and more strongly the A-site of 70S ribosomes. We suggest that EF-P: (a) may play a role in translational fidelity and (b) prevents entry of fMet-tRNA into the A-site enabling it to bind to the 50S P-site. We also report that EF-P promotes a ribosome-dependent accommodation of fMet-tRNA into the 70S P-site.
Large-scale proteomic analyses in Escherichia coli have documented the composition and physical r... more Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among cproteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.
SUMMARY Current X-ray diffraction and cryoelectron microscopic data of ribosomes of eubacteria ha... more SUMMARY Current X-ray diffraction and cryoelectron microscopic data of ribosomes of eubacteria have shed considerable light on the molecular mechanisms of translation. Structural studies of the protein factors that activate ribosomes also point to many common features in the primary sequence and tertiary structure of these proteins. The reconstitution of the complex apparatus of translation has also revealed new information important to the mechanisms. Surprisingly, the latter approach has uncovered a number of proteins whose sequence and/or structure and function are conserved in all cells, indicating that the mechanisms are indeed conserved. The possible mechanisms of a new initiation factor and two elongation factors are discussed in this context.
A key component in determining the functional role of any protein is the elucidation of its bindi... more A key component in determining the functional role of any protein is the elucidation of its binding partners using protein-protein interaction (PPI) data. Here we examine the use of tandem affinity purification (TAP) tagging to study RNA/DNA helicase PPIs in Escherichia coli. The tag, which consists of a calmodulin-binding region, a TEV protease recognition sequence, and an IgG-binding domain, is introduced into E. coli using a lambdared recombination system. This method prevents the overproduction of the target protein, which could generate false interactions. The interacting proteins are then affinity purified using double affinity purification steps and are separated by SDS-PAGE followed by mass spectrometry identification. Each protein identified would represent a physical interaction in the cell. These interactions may potentially be mediated by an RNA/DNA template, for which the helicase would likely be needed to disrupt the secondary structures.
The in vitro reversal of conditionally lethal mutations has greatly aided the study of translatio... more The in vitro reversal of conditionally lethal mutations has greatly aided the study of translation. N4316 is a mutant of Escherichia coli that has a temperature-sensitive defect in a protein called the rescue protein. Without the rescue protein, translation in vivo and in vitro is drastically reduced and frameshift errors, as well as increased read-through of nonsense codons, occurs. Using reversal of temperature-sensitivity as an assay, the rescue protein was purified from a ribosomal eluate of the parental (D10) strain. Composite polyacrylamide/agarose gel electrophoresis and sedimentation on sucrose density gradients were employed to examine the distribution of 70s ribosomes and ribosomal subunits in the mutant (N4316) and the parental (D10) extracts at restrictive (43°C) and non-restrictive (35 "C) temperatures. Fewer polysomes and a larger proportion of 70s ribosomes relative to subunits were observed at 43°C with N4316, but not with D10 extracts. Addition of the rescue protein had no effect at 35°C with either strain, but restored the polysome pattern of N4316 at 43°C. The purified rescue protein labelled by methylation retained activity and bound preferentially to 30s subunits. Rescue bound to 30s particles prevented the action of IF-3 fostering formation of 70s ribosomes. Thus the rescue protein enables formation of 70s ribosomes from 30s and 50s subunits. 70s ribosomes which contain the rescue protein are active in translation and resist dissociation induced by high centrifugal fields. We propose that the rescue protein alters the conformation of 70s ribosomes resulting in a tighter association of subunits which, in turn, fosters both higher rates and increased accuracy of translation. Conditionally lethal mutants have been extremely useful in studies of the mechanisms underlying translation [ 1-31. One such mutant is strain N4316 which is derived from the Escherichia coli thymidine auxotroph D10 (met-, thy-, RNase I-). N4316 was isolated by a procedure designed to optimize the selection for temperature-sensitive ribosomal mutants [4, 51. At 43"C, translation in vivo is impaired and N4316 cells die over a period of a few hours [4]. At 36"C, N4316 suppresses termination codons UGA and UAA, and occasionally UAG, as well as certain missense codons [4, 51. The temperature-sensitive growth and temperature-dependent suppression are believed to be due to lesions in two separate genes (sts and sut, respectively) which map at different chromosomal loci and can be separated by transduction and cloning [6-81. At restrictive temperatures [4], crude extracts of
Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translatio... more Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translation elongation in some lower fungi. Here we report the first identification of a ribosome bound ATPase, RbbA, in E. coli cells. RbbA accounts for most of the ATPase activity associated with 70S ribosomes and 30S ribosomal subunits. Both native and recombinant RbbA were purified and shown to possess ribosome-dependent ATPase activities and to stimulate polyphenylalanine synthesis in vitro. Biochemically, RbbA is similar to the fungi-specific translation elongation factor 3 (EF-3) and cross-reacts with antibody raised against EF-3. The gene encoding RbbA is identified as ORF yhih and the predicted RbbA amino acid sequence is 40% similar to that of the C-terminal half of EF-3. The discovery of a ribosomal ATPase in a prokaryotic cell suggests a common, conserved function for these proteins in translation.
Biochemical and Biophysical Research Communications, 2005
The oxazolidinones are a new class of potent antibiotics that are active against a broad spectrum... more The oxazolidinones are a new class of potent antibiotics that are active against a broad spectrum of Gram-positive bacterial pathogens including those resistant to other antibiotics. These drugs specifically inhibit protein biosynthesis whereas DNA and RNA synthesis are not affected. Although biochemical and genetic studies indicate that oxazolidinones target the ribosomal peptidyltransferase center, other investigations suggest that they interact with different regions of ribosomes. Thus, the exact binding site and mechanism of action have remained elusive. Here, we study, by use of base-specific reagents, the effect of the oxazolidinones on the chemical protection footprinting patterns of the 23S rRNA. We report: (i) reproducible protection of G2607 and G2608 of 23S rRNA by a potent oxazolidinone on a ribosomeAEtRNAAEmRNA complex; (ii) no protections were observed on 70S ribosomes devoid of tRNA and mRNA; (iii) EF-G also weakly protected G2607 and G2608; (iv) mutations at G2608 conferred resistance to the oxazolidinones in Escherichia coli cells; and (v) G2607 and G2608 occur near the exit to the peptide tunnel on the 50S subunit. A mechanism for the pleiotropic action of the oxazolidinones is discussed.
Bacillus amyloliquefaciens strain AL 35, produced high yields of a cyclodextrin glycosyltransfera... more Bacillus amyloliquefaciens strain AL 35, produced high yields of a cyclodextrin glycosyltransferase (CGT'ase) when grown in a submerged culture. The stability of CGT'ase to high temperature and alkaline pH enabled processing for cyclodextrin production to be carried out at 60°C and pH 9.0. Crude culture filtrates containing the CGT'ase could convert gelatinized starch substrates to predominantly a-cyclodextrins (up to 95% of the total cyclodextrin yields).
The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine–threonine kinase, en... more The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine–threonine kinase, encompassing three catalytic (Tpk1–3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the tpk1 catalytic subunit reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ factor (nej1), co-function in the same pathway, and parallel to the NHEJ factor yku80. Chromatin immunoprecipitation and resection data suggest that tpk1 deletion influences repair protein recruitments and DNA resection. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair and nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKA...
2-Methylcitrate dehydratase (2-methylcitrate hydro-lyase) functioning at the methylcitric acid cy... more 2-Methylcitrate dehydratase (2-methylcitrate hydro-lyase) functioning at the methylcitric acid cycle of propionyl-CoA oxidation was purified from a cell-free extract of Yarrowia
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1987
l. fl-N-acetylglucosaminidase and one of the several chitinases were purified from the supernatan... more l. fl-N-acetylglucosaminidase and one of the several chitinases were purified from the supernatant fraction from homogenates of pupae of the red flour beetle, Tribolium castaneum Herbst, by ammonium sulfate fractionation, hydroxylapatite chromatography, anion exchange chromatography and gel filtration chromatography. 2. //-N-acetylglucosaminidase exhibited a dimeric structure with apparent subunit molecular weights of 7.3 x 104 and 6.4 x 104, whereas chitinase was a monomer with an apparent mol. wt of 7.7 x 104. 3. //-N-acetylglucosaminidase and chitinase were composed of approximately 1200 and 700 amino acid residues per molecule and exhibited isoelectric points of pH 4.9 and 4.7, respectively. 4. The kcat/K m value for fl-N-acetylglucosaminidase hydrolyzing flGIcNAc 2 was 2.22 x 104 M-I see-I while that for chitinase hydrolyzing glycol chitin was 6.4 rnl mg-l see-t. 5. The results demonstrate a chitinolytic enzyme system in 7". castaneum composed of an endo-splitting chitinase and an exo-splitting fl-N-acetylglucosaminidase. *Contribution No. 86-324-J. Department of Biochemistry, Kansas Agricultural Experiment Station, Manhattan, Kansas 66506. Cooperative investigation between ARS, USDA and the Kansas Agricultural Experiment Station. Mention of a proprietary product does not imply approval of this product by the USDA to the exclusion of other products that may also be suitable.
Disruption of eshA , which encodes a 52-kDa protein that is produced late during the growth of St... more Disruption of eshA , which encodes a 52-kDa protein that is produced late during the growth of Streptomyces coelicolor A3(2), resulted in elimination of actinorhodin production. In contrast, disruption of eshB , a close homologue of eshA , had no effect on antibiotic production. The eshA disruptant accumulated lower levels of ppGpp than the wild-type strain accumulated. The loss of actinorhodin production in the eshA disruptant was restored by expression of a truncated relA gene, which increased the ppGpp level to the level in the wild-type strain, indicating that the reduced ppGpp accumulation in the eshA mutant was solely responsible for the loss of antibiotic production. Antibiotic production was also restored in the eshA mutant by introducing mutations into rpoB (encoding the RNA polymerase β subunit) that bypassed the requirement for ppGpp, which is consistent with a role for EshA in modulating ppGpp levels. EshA contains a cyclic nucleotide-binding domain that is essential for...
Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of ... more Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus . Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent ( rel + ; wild type) and relaxed ( relA and relC ; mutant) strains of T. thermophilus . We found that in wild-type T. thermophilus , as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA Ser aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA -null mutant and partially blocked in a relC mutant harboring a muta...
We have previously isolated and described an Escherichia coli ribosome-bound ATPase, RbbA, that i... more We have previously isolated and described an Escherichia coli ribosome-bound ATPase, RbbA, that is required for protein synthesis in the presence of ATP, GTP and the elongation factors, EF-Tu and EF-G. The gene encoding RbbA, yhih, has been cloned and the deduced protein sequence harbors two ATP-motifs and one RNA-binding motif and is homologous to the fungal EF-3. Here, we describe the isolation and assay of a truncated form of the RbbA protein that is stable to overproduction and purification. Chemical protection results show that the truncated RbbA specifically protects nucleotide A937 on the 30S subunit of ribosomes, and the protected site occurs at the E-site where the tRNA is ejected upon A-site occupation. Other weakly protected bases in the region occur at or near the mRNA binding site. Using radiolabeled tRNAs, we study the stimulating effect of this truncated RbbA on the binding and release of different tRNAs bound to the (aminoacyl) A-, (peptidyl) P-and (exit) E-sites of 70S ribosomes. The combined data suggest plausible mechanisms for the function of RbbA in translation.
The oxazolidinones represent a new class of antimicrobial agents which are active against multidr... more The oxazolidinones represent a new class of antimicrobial agents which are active against multidrug-resistant staphylococci, streptococci, and enterococci. Previous studies have demonstrated that oxazolidinones inhibit bacterial translation in vitro at a step preceding elongation but after the charging of N -formylmethionine to the initiator tRNA molecule. The event that occurs between these two steps is termed initiation. Initiation of protein synthesis requires the simultaneous presence of N -formylmethionine-tRNA, the 30S ribosomal subunit, mRNA, GTP, and the initiation factors IF1, IF2, and IF3. An initiation complex assay measuring the binding of [ 3 H] N -formylmethionyl-tRNA to ribosomes in response to mRNA binding was used in order to investigate the mechanism of oxazolidinone action. Linezolid inhibited initiation complex formation with either the 30S or the 70S ribosomal subunits from Escherichia coli . In addition, complex formation with Staphylococcus aureus 70S tight-co...
G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with ke... more G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable re...
Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play cru... more Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provide...
Bacterial protein synthesis is an essential, conserved, and environmentally responsive process. Y... more Bacterial protein synthesis is an essential, conserved, and environmentally responsive process. Yet, many of its components and dependencies remain unidentified. To address this gap, we used quantitative synthetic genetic arrays to map functional relationships among >48,000 gene pairs in Escherichia coli under four culture conditions differing in temperature and nutrient availability. The resulting data provide global functional insights into the roles and associations of genes, pathways, and processes important for efficient translation, growth, and environmental adaptation. We predict and independently verify the requirement of unannotated genes for normal translation, including a previously unappreciated role of YhbY in 30S biogenesis. Dynamic changes in the patterns of genetic dependencies across the four growth conditions and data projections onto other species reveal overarching functional and evolutionary pressures impacting the translation system and bacterial fitness, un...
Highlights d Analyses of ubiquitous protein complexes identified new components in ASD d HDAC1/2 ... more Highlights d Analyses of ubiquitous protein complexes identified new components in ASD d HDAC1/2 positively regulates ASD orthologs in the mouse embryonic brain d IP/MS in neuronal cells identified protein complexes in ASD d A network bridges the gap between the idiopathic and syndromic forms of ASD
The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrit... more The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast ( Saccharomyces cerevisiae ) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identifie...
EF-P (eubacterial elongation factor P) is a highly conserved protein essential for protein synthe... more EF-P (eubacterial elongation factor P) is a highly conserved protein essential for protein synthesis. We report that EF-P protects 16S rRNA near the G526 streptomycin and the S12 and mRNA binding sites (30S T-site). EF-P also protects domain V of the 23S rRNA proximal to the A-site (50S T-site) and more strongly the A-site of 70S ribosomes. We suggest that EF-P: (a) may play a role in translational fidelity and (b) prevents entry of fMet-tRNA into the A-site enabling it to bind to the 50S P-site. We also report that EF-P promotes a ribosome-dependent accommodation of fMet-tRNA into the 70S P-site.
Large-scale proteomic analyses in Escherichia coli have documented the composition and physical r... more Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among cproteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.
SUMMARY Current X-ray diffraction and cryoelectron microscopic data of ribosomes of eubacteria ha... more SUMMARY Current X-ray diffraction and cryoelectron microscopic data of ribosomes of eubacteria have shed considerable light on the molecular mechanisms of translation. Structural studies of the protein factors that activate ribosomes also point to many common features in the primary sequence and tertiary structure of these proteins. The reconstitution of the complex apparatus of translation has also revealed new information important to the mechanisms. Surprisingly, the latter approach has uncovered a number of proteins whose sequence and/or structure and function are conserved in all cells, indicating that the mechanisms are indeed conserved. The possible mechanisms of a new initiation factor and two elongation factors are discussed in this context.
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Papers by Hiroyuki Aoki