Biomedical subjects

W S Lane

Publications and source records attributed to W S Lane.

At least 73 records · Page 4Linked to original sources

PubMed1998

The 20kD protein of human [U4/U6.U5] tri-snRNPs is a novel cyclophilin that forms a complex with the U4/U6-specific 60kD and 90kD proteins.

Cyclophilins (Cyps) catalyze the cis/trans isomerization of peptidyl-prolyl bonds, a rate-limiting step in protein folding. In some cases, cyclophilins have also been shown to form stable complexes with specific proteins in vivo and may thus also act as chaperone-like molecules. We have characterized the 20kD protein of the spliceosomal 25S [U4/U6.U5] tri-snRNP complex from HeLa cells and show that it is a novel human cyclophilin (denoted SnuCyp-20). Purified [U4/U6.U5] tri-snRNPs, but not U1, U2, or U5 snRNPs, exhibit peptidyl-prolyl cis/trans isomerase activity in vitro, which is cyclosporin A-sensitive, suggesting that SnuCyp-20 is an active isomerase. Consistent with its specific association with tri-snRNPs in vitro, immunofluorescence microscopy studies showed that SnuCyp-20 is predominantly located in the nucleus, where it colocalizes in situ with typical snRNP-containing structures referred to as nuclear speckles. As a first step toward the identification of possible targets of SnuCyp-20, we have investigated the interaction of SnuCyp-20 with other proteins of the tri-snRNP. Fractionation of RNA-free protein complexes dissociated from isolated tri-snRNPs by treatment with high salt revealed that SnuCyp-20 is part of a biochemically stable heteromer containing additionally the U4/U6-specific 60kD and 90kD proteins. By coimmunoprecipitation experiments performed with in vitro-translated proteins, we could further demonstrate a direct interaction between SnuCyp-20 and the 60kD protein, but failed to detect a protein complex containing the 90kD protein. The formation of a stable SnuCyp-20/60kD/90kD heteromer may thus require additional factors not present in our in vitro reconstitution system. We discuss possible roles of SnuCyp-20 in the assembly of [U4/U6.U5] tri-snRNPs and/or in conformational changes occurring during the splicing process.

Amino Acid Sequence↗

PubMed1997

A novel 160-kDa phosphotyrosine protein in insulin-treated embryonic kidney cells is a new member of the insulin receptor substrate family.

We have previously identified a 160-kDa protein in human embryonic kidney (HEK) 293 cells that undergoes rapid tyrosine phosphorylation in response to insulin (PY160) (Kuhné, M. R., Zhao, Z., and Lienhard, G. E. (1995) Biochem. Biophys. Res. Commun. 211, 190-197). The phosphotyrosine form of PY160 was purified from insulin-treated HEK 293 cells by anti-phosphotyrosine immunoaffinity chromatography, the sequences of peptides determined, and its cDNA cloned. The PY160 cDNA encodes a 1257-amino acid protein that contains, in order from its N terminus, a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain, and, spread over the C-terminal portion, 12 potential tyrosine phosphorylation sites. Several of these sites are in motifs expected to bind specific SH2 domain-containing proteins: YXXM (7 sites), phosphatidylinositol 3-kinase; YVNM (1 site), Grb-2; and YIEV (1 site), either the protein-tyrosine phosphatase SHP-2 or phospholipase Cgamma. Furthermore, the PH and PTB domains are highly homologous (at least 40% identical) to those found in insulin receptor substrates 1, 2, and 3 (IRS-1, IRS-2, and IRS-3). Thus, PY160 is a new member of the IRS family, which we have designated IRS-4.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase.

Phosphatidylinositol 3-kinases (PI 3-kinases) have been implicated in membrane trafficking in the secretory and endocytic pathways of yeast and mammalian cells, but the molecular mechanisms by which these lipid kinases operate are not known. Here we identify a protein of 170 kDa that is rapidly released from cell membranes in response to wortmannin, a potent inhibitor of mammalian PI 3-kinases. The amino acid sequence of peptides from p170 reveal its identity to early endosomal antigen (EEA) 1, an endosomal antigen with homology to several yeast proteins genetically implicated in membrane trafficking. Immunofluorescence analysis of 3T3-L1 adipocytes with antisera against p170/EEA1 reveal a punctate peripheral pattern that becomes diffuse in response to wortmannin. In vitro, p170/EEA1 binds specifically to liposomes containing PIns(3)P, suggesting that the effect of wortmannin on cells is due to inhibition of PIns(3)P production. Thus, p170/EEA1 may define a family of proteins that mediate the regulatory effects of 3'-phosphoinositides on membrane trafficking in yeast and mammalian cells.

3T3 Cells↗

PubMed1997

An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2.

The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5-116kD) that is strikingly homologous to the ribosomal elongation factor EF-2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5-116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre-mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5-116kD inhibit pre-mRNA splicing in a HeLa nuclear extract in vitro. In HeLa cells, U5-116kD is located in the nucleus and colocalizes with snRNP-containing subnuclear structures referred to as speckles. The G domain of U5-116kD/Snu114p contains the consensus sequence elements G1-G5 important for binding and hydrolyzing GTP. Consistent with this, U5-116kD can be cross-linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP-binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5-116kD/Snu114p. This is to date the first evidence that a G domain-containing protein plays an essential role in the pre-mRNA splicing process.

Amino Acid Sequence↗

PubMed1997

Purification of a Tat-associated kinase reveals a TFIIH complex that modulates HIV-1 transcription.

The Tat protein is a transcriptional activator which is required for efficient human immunodeficiency virus 1 (HIV-1) gene expression Tat stimulates HIV-1 transcriptional elongation by increasing the processivity of RNA polymerase II. To address whether Tat-mediated effects on HIV-1 gene expression are due to modulation in the phosphorylation of the RNA polymerase II C-terminal domain (CTD), we developed a purification protocol to identify cellular kinases that are capable of binding to Tat and hyperphosphorylating the RNA polymerase II CTD. A 600 kDa protein complex with these properties was isolated, and specific components were identified using peptide microsequence analysis. This analysis indicated that proteins comprising the multi-subunit TFIIH complex, in addition to several novel factors, were associated with Tat using both in vitro and in vivo analysis. The Tat-associated kinase bound to the activation domain of Tat, and its ability to hyperphosphorylate RNA polymerase II was markedly stimulated by Tat. Furthermore, the addition of the Tat-associated kinase to in vitro transcription assays stimulated the ability of Tat to activate HIV-1 transcription. These results define a cellular kinase complex whose activity is modulated by Tat to result in activation of HIV-1 trancription.

Cyclin-Dependent Kinases↗

PubMed1997

The 60-kDa phosphotyrosine protein in insulin-treated adipocytes is a new member of the insulin receptor substrate family.

A 60-kDa protein that undergoes rapid tyrosine phosphorylation in response to insulin and then binds phosphatidylinositol 3-kinase has been previously described in adipocytes and hepatoma cells. We have isolated this protein, referred to as pp60, from rat adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA. The predicted amino acid sequence of pp60 reveals that it contains an N-terminal pleckstrin homology domain, followed by a phosphotyrosine binding domain, followed by a group of likely tyrosine phosphorylation sites, four of which are in the YXXM motif that binds to the SH2 domains of phosphatidylinositol 3-kinase. The overall architecture of pp60 is thus the same as that of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), and furthermore both the pleckstrin homology and phosphotyrosine binding domains are highly homologous (about 50% identical amino acids) to these domains in both IRS-1 and IRS-2. Thus, pp60 is a new member of the IRS family, which we have designated IRS-3.

Adipocytes↗

PubMed1997

Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.

We previously identified the angiogenesis inhibitor angiostatin. Using a similar strategy, we have identified endostatin, an angiogenesis inhibitor produced by hemangioendothelioma. Endostatin is a 20 kDa C-terminal fragment of collagen XVIII. Endostatin specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth. By a novel method of sustained release, E. coli-derived endostatin was administered as a nonrefolded suspension. Primary tumors were regressed to dormant microscopic lesions. Immunohistochemistry revealed blocked angiogenesis accompanied by high proliferation balanced by apoptosis in tumor cells. There was no toxicity. Together with angiostatin data, these findings validate a strategy for identifying endogenous angiogenesis inhibitors, suggest a theme of fragments of proteins as angiogenesis inhibitors, and demonstrate dormancy therapy.

Amino Acid Sequence↗

PubMed1997

The critical active-site amine of the human 8-oxoguanine DNA glycosylase, hOgg1: direct identification, ablation and chemical reconstitution.

BACKGROUND: Base-excision DNA repair (BER) is the principal pathway responsible for the removal of aberrant, genotoxic bases from the genome and restoration of the original sequence. Key components of the BER pathway are DNA glycosylases, enzymes that recognize aberrant bases in the genome and catalyze their expulsion. One major class of such enzymes, glycosylase/lyases, also catalyze scission of the DNA backbone following base expulsion. Recent studies indicate that the glycosylase and lyase functions of these enzymes are mechanistically unified through a common amine-bearing residue on the enzyme, which acts as both the electrophile that displaces the aberrant base and an electron sink that facilitates DNA strand scission through imine (Schiff base)/conjugate elimination chemistry. The identity of this critical amine-bearing residue has not been rigorously established for any member of a superfamily of BER glycosylase/lyases. RESULTS: Here, we report the identification of the active-site amine of the human 8-oxoguanine DNA glycosylase (hOgg1), a human BER superfamily protein that repairs the mutagenic 8-oxoguanine lesion in DNA. We employed Edman sequencing of an active-site peptide irreversibly linked to substrate DNA to identify directly the active-site amine of hOgg1 as the epsilon-NH2 group of Lys249. In addition, we observed that the repair-inactive but recognition-competent Cys249 mutant (Lys249-->Cys) of hOgg1 can be functionally rescued by alkylation with 2-bromoethylamine, which functionally replaces the lysine residue by generating a gamma-thia-lysine. CONCLUSIONS: This study provides the first direct identification of the active-site amine for any DNA glycosylase/lyase belonging to the BER superfamily, members of which are characterized by the presence of a helix-hairpin-helix-Gly/Pro-Asp active-site motif. The critical lysine residue identified here is conserved in all members of the BER superfamily that exhibit robust glycosylase/lyase activity. The ability to trigger the catalytic activity of the Lys249-->Cys mutant of hOgg1 by treatment with the chemical inducer 2-bromoethylamine may permit snapshots to be taken of the enzyme acting on its substrate and could represent a novel strategy for conditional activation of catalysis by hOgg1 in cells.

Amines↗

PubMed1997

CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in Tat-activated human immunodeficiency virus type 1 transcription.

Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme. Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150.

Alanine↗

PubMed1997

The human U4/U6 snRNP contains 60 and 90kD proteins that are structurally homologous to the yeast splicing factors Prp4p and Prp3p.

Immunoaffinity-purified human 25S [U4/U6.U5] tri-snRNPs harbor a set of polypeptides, termed the tri-snRNP proteins, that are not present in Mono Q-purified 20S U5 snRNPs or 10S U4/U6 snRNPs and that are important for tri-snRNP complex formation (Behrens SE, Lührmann R, 1991, Genes & Dev 5:1439-1452). Biochemical and immunological characterization of HeLa [U4/U6.U5] tri-snRNPs led to the identification of two novel proteins with molecular weights of 61 and 63kD that are distinct from the previously described 15.5, 20, 27, 60, and 90kD tri-snRNP proteins. For the initial characterization of tri-snRNP proteins that interact directly with U4/U6 snRNPs, immunoaffinity chromatography with an antibody directed against the 60kD protein was performed. We demonstrate that the 60 and 90kD tri-snRNP proteins specifically associate with the U4/U6 snRNP at salt concentrations where the tri-snRNP complex has dissociated. The primary structures of the 60kD and 90kD proteins were determined by cloning and sequencing their respective cDNAs. The U4/U6-60kD protein possesses a C-terminal WD domain that contains seven WD repeats and thus belongs to the WD-protein family, whose best-characterized members include the Gbeta subunits of heterotrimeric G proteins. A database homology search revealed a significant degree of overall homology (57.8% similarity, 33.9% identity) between the human 60kD protein and the Saccharomyces cerevisiae U4/U6 snRNP protein Prp4p. Two additional, previously undetected WD repeats (with seven in total) were also identified in Prp4p, consistent with the possibility that 60kD/Prp4p, like beta-transducin, may adopt a propeller-like structure. The U4/U6-90kD protein was shown to exhibit significant homology, particularly in its C-terminal half, with the S. cerevisiae splicing factor Prp3p, which also associates with the yeast U4/U6 snRNP. Interestingly, U4/U6-90kD shares short regions of homology with E. coli RNase III, including a region encompassing its double-stranded RNA binding domain. Based on their structural similarity with essential splicing factors in yeast, the human U4/U6-60kD and 90kD proteins are likely also to play important roles in the mammalian splicing process.

Amino Acid Sequence↗

PubMed1996

Covalent HLA-B27/peptide complex induced by specific recognition of an aziridine mimic of arginine.

The class I major histocompatibility complex (MHC) glycoprotein HLA-B27 binds short peptides containing arginine at peptide position 2 (P2). The HLA-B27/peptide complex is recognized by T cells both as part of the development of the repertoire of T cells in the cellular immune system and during activation of cytotoxic T cells. Based on the three-dimensional structure of HLA-B27, we have synthesized a ligand with an aziridine-containing side chain designed to mimic arginine and to bind covalently in the arginine-specific P2 pocket of HLA-B27. Using tryptic digestion followed by mass spectrometry and amino acid sequencing, the aziridine-containing ligand is shown to alkylate specifically cysteine 67 of HLA-B27. Neither free cysteine in solution nor an exposed cysteine on a class II MHC molecule can be alkylated, showing that specific recognition between the anchor side-chain pocket of an MHC class I protein and the designed ligand (propinquity) is necessary to induce the selective covalent reaction with the MHC class I molecule.

Affinity Labels↗

PubMed1996

Replication protein A is a component of a complex that binds the human metallothionein IIA gene transcription start site.

Previous studies revealed that sequences surrounding the initiation sites in many mammalian and viral gene promoters, called initiator (Inr) elements, may be essential for promoter strength and for determining the actual transcription start sites. DNA sequences in the vicinity of the human metallothionein IIA (hMTIIA) gene transcription start site share homology with some of the previously identified Inr elements. However, in the present study we have found by in vitro transcription assays that the hMTIIA promoter does not contain a typical Inr. Electrophoretic mobility shift assays identified several DNA-protein complexes at the hMTIIA gene transcription start site. A partially purified protein fraction containing replication protein A (RPA) binds to the hMTIIA gene transcription start site and represses transcription from the hMTIIA promoter in vitro. In addition, overexpression of the human 70-kDa RPA-1 protein represses transcription of a reporter gene controlled by the hMTIIA promoter in vivo. These findings suggest that hMTIIA transcription initiation is controlled by a mechanism different from most mammalian and viral promoters and that the previously identified RPA may also be involved in transcription regulation.

Amino Acid Sequence↗

PubMed1996

Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily.

BACKGROUND: Reactive oxygen species, ionizing radiation, and other free radical generators initiate the conversion of guanine (G) residues in DNA to 8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with adenine (A) during replication. Bacteria counter this threat with a multicomponent system that excises the lesion, corrects OG:A mispairs and cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence has suggested the existence of base-excision DNA repair proteins specific for OG in eukaryotes, little is known about these proteins. RESULTS: Using substrate-mimetic affinity chromatography followed by a mechanism-based covalent trapping procedure, we have isolated a base-excision DNA repair protein from Saccharomyces cerevisiae that processes OG opposite cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome database using peptide sequences from the protein identified a gene, OGG1, encoding a predicted 43 kDa (376 amino acid) protein, identical to one identified independently by complementation cloning. Ogg1 has OG:C-specific base-excision DNA repair activity and also intrinsic beta-lyase activity, which proceeds through a Schiff base intermediate. Targeted disruption of the OGG1 gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G. CONCLUSIONS: S. cerevisiae has two OG-specific glycosylase/lyases, which differ significantly in their preference for the base opposite the lesion. We suggest that one of these, Ogg1, is closely related in overall three-dimensional structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase that acts on fragmented and oxidatively damaged pyrimidines. We have recently shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases, is structurally homologous to endo III. We have now identified a shared active site motif amongst these three proteins. Using this motif as a protein database searching tool, we find that it is present in a number of other base-excision DNA repair proteins that process diverse lesions. Thus, we propose the existence of a DNA glycosylase superfamily, members of which possess a common fold yet act upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to alkylated or oxidized bases.

Amino Acid Sequence↗

PubMed1996

The HeLa 200 kDa U5 snRNP-specific protein and its homologue in Saccharomyces cerevisiae are members of the DEXH-box protein family of putative RNA helicases.

The primary structure of the 200 kDa protein of purified HeLa U5 snRNPs (U5-200kD) was characterized by cloning and sequencing of its cDNA. In order to confirm that U5-200kD is distinct from U5-220kD we demonstrate by protein sequencing that the human U5-specific 220 kDa protein is homologous to the yeast U5-specific protein Prp8p. A 246 kDa protein (Snu246p) homologous to U5-200kD was identified in Saccharomyces cerevisiae. Both proteins contain two conserved domains characteristic of the DEXH-box protein family of putative RNA helicases and RNA-stimulated ATPases. Antibodies raised against fusion proteins produced from fragments of the cloned mammalian cDNA interact specifically with the HeLa U5-200kD protein on Western blots and co-immunoprecipitate U5 snRNA and to a lesser extent U4 and U6 snRNAs from HeLa snRNPs. Similarly, U4, U5 and U6 snRNAs can be co-immunoprecipitated from yeast splicing extracts containing an HA-tagged derivative of Snu246p with HA-tag specific antibodies. U5-200kD and Snu246p are thus the first putative RNA helicases shown to be intrinsic components of snRNPs. Disruption of the SNU246 gene in yeast is lethal and leads to a splicing defect in vivo, indicating that the protein is essential for splicing. Anti-U5-200kD antibodies specifically block the second step of mammalian splicing in vitro, demonstrating for the first time that a DEXH-box protein is involved in mammalian splicing. We propose that U5-200kD and Snu246p promote one or more conformational changes in the dynamic network of RNA-RNA interactions in the spliceosome.

Amino Acid Sequence↗

PubMed1996

Cloning and functional characterization of the gene encoding the TFIIIB90 subunit of RNA polymerase III transcription factor TFIIIB.

The yeast RNA polymerase III (pol III) general transcription factor TFIIIB is composed of three subunits; the TATA-binding protein (TBP)1, the TFIIB-related factor (BRF1), and a third factor termed TFIIIB90 or B". Here we report the purification of yeast TFIIIB90, cloning of the gene encoding TFIIIB90, and reconstitution of TFIIIB from recombinant polypeptides. The TFIIIB90 open reading frame encodes a 68-kDa polypeptide and has no obvious similarity to any other known protein sequences. The gene encoding TFIIIB90 is essential for viability of yeast. Using recombinant TFIIIB subunits, we found that TFIIIB90 interacts weakly with TBP in the absence of BRF1, and that this interaction is enhanced at least 25-fold by BRF1. In addition, TFIIIB90 showed pol III specificity as it could not interact with the pol II-specific TFIIB-TBP-DNA complex. To localize the regions of the TBP-DNA complex that interact with BRF1 and TFIIIB90, we tested whether the pol II factors TFIIA and TFIIB interfered with the binding of BRF1 and TFIIIB90 to TBP-DNA. Our results suggest that the binding sites for BRF1 and TFIIIB90 on TBP-DNA both overlap the binding sites for TFIIA and TFIIB.

Amino Acid Sequence↗

PubMed1996

Didemnin binds to the protein palmitoyl thioesterase responsible for infantile neuronal ceroid lipofuscinosis.

The marine natural product didemnin B, currently in clinical trials as an antitumor agent, has several potent biological activities apparently mediated by distinct mechanisms. Our initial investigation of didemnin B resulted in the discovery of its GTP-dependent binding of the translation elongation factor EF1 alpha. This finding is consistent with the protein synthesis inhibitory activity of didemnin B observed at intermediate concentrations. To begin to dissect the mechanisms involved in the cytostatic and immunosuppressive activities of didemnin B, observed at low concentrations, additional didemnin-binding proteins were sought. Here we report the purification of a 36-kDa glycosylated didemnin-binding protein from bovine brain lysate. Cloning of the human cDNA encoding this protein revealed a strong sequence similarity with palmitoyl protein thioesterase (PPT), an enzyme that removes palmitate from H-Ras and the G alpha s subunits of heterotrimeric GTP-binding proteins in vitro. Mutations in PPT have recently been shown to be responsible for infantile neuronal ceroid lipofuscinosis, which is a severe brain disorder characterized by progressive loss of brain function and early death.

Amino Acid Sequence↗

PubMed1996

Characterization of a Cys115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-GlcNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin.

The antibiotic fosfomycin inhibits bacterial cell wall biosynthesis by inactivation of UDP-GlcNAc enolpyruvyl tranferase (MurA). Prior work has established that Cys115 of Escherichia coli and Enterobacter cloacae MurA is the active site nucleophile alkylated by fosfomycin and implicated this residue in the formation of a covalent phospholactyl-enzyme adduct derived from substrate, phosphoenolpyruvate (PEP). On the basis of sequencing information from putative MurA homolog from Mycobacterium tuberculosis, we generated a C115D mutant of E. coli MurA that was highly active but fully resistant to time-dependent inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition vs PEP. In contrast to the broad pH-independent behavior of wild-type (WT) MurA, C115D mutant activity titrated across the pH range examined (pH 5.5-9) with an apparent pKa approximately 6, with kcatC115D ranging from approximately 10kcatWT at pH 5.5 to <0.1kcatWT at pH9.0. Km(PEP)115D was relatively constant in the pH range examined and increased approximately 100-fold relative to Km(PEP)WT. A fosfomycin-resistant C115E mutant with -1% activity of the C115D mutant was found to follow a pH dependence similar to that observed for C115D MurA. The contrasting pH dependences of WT and C115D MurA was also observed in the reaction with the pseudosubstrate, (Z)-3-fluorophosphoenolpyruvate, strongly suggesting a role for Cys/Asp115 as the general acid in the protonation of C-3 of PEP during MurA-catalyzed enol ether transfer. The difference in nucleophilicity between the carboxylate side chains of Asp115 and Glu115 and the thiolate group of Cys115 suggests that covalent enzyme adduct formation is not required for catalytic turnover and, furthermore, provides a chemical rationale for the resistance of the C115D and C115E mutants to fosfomycin inactivation.

Alkyl and Aryl Transferases↗

PubMed1996

An RNA polymerase II elongation factor encoded by the human ELL gene.

The human ELL gene on chromosome 19 undergoes frequent translocations with the trithorax-like MLL gene on chromosome 11 in acute myeloid leukemias. Here, ELL was shown to encode a previously uncharacterized elongation factor that can increase the catalytic rate of RNA polymerase II transcription by suppressing transient pausing by polymerase at multiple sites along the DNA. Functionally, ELL resembles Elongin (SIII), a transcription elongation factor regulated by the product of the von Hippel-Lindau (VHL) tumor suppressor gene. The discovery of a second elongation factor implicated in oncogenesis provides further support for a close connection between the regulation of transcription elongation and cell growth.

Amino Acid Sequence↗