Search

TFIIB is essential for transcription initiation by RNA polymerase II. TFIIB also cross-links to terminator regions and is required for gene loops that juxtapose promoter-terminator elements in a transcription-dependent manner. The Saccharomyces cerevisiae sua7-1 mutation encodes an altered form of TFIIB (E62K) that is defective for both start site selection and gene looping. Here we report the isolation of an ssl2 mutant, encoding an altered form of TFIIH, as a suppressor of the cold-sensitive growth defect of the sua7-1 mutation. Ssl2 (Rad25) is orthologous to human XPB and is a member of the SF2 family of ATP-dependent DNA helicases. The ssl2 suppressor allele encodes an arginine replacement of the conserved histidine residue (H508R) located within the DEVH-containing helicase domain. In addition to suppressing the TFIIB E62K growth defect, Ssl2 H508R partially restores both normal start site selection and gene looping. Moreover, Ssl2, like TFIIB, associates with promoter and terminator regions, and the diminished association of TFIIB E62K with the PMA1 terminator is restored by the Ssl2 H508R suppressor. These results define a novel, functional interaction between TFIIB and Ssl2 that affects start site selection and gene looping.

We report herein a critical role for the stringent response regulatory DnaK suppressor protein (DksA) in the coordination of antioxidant defenses. DksA helps fine-tune the expression of glutathione biosynthetic genes and discrete steps in the pentose phosphate pathway and tricarboxylic acid cycle that are associated with the generation of reducing power. Control of NAD(P)H/NAD(P)(+) redox balance by DksA fuels downstream antioxidant enzymatic systems in nutritionally starving Salmonella. Conditional expression of the glucose-6-phosphate dehydrogenase-encoding gene zwf, shown here to be under DksA control, increases both the NADPH pool and antioxidant defenses of dksA mutant Salmonella. The DksA-mediated coordination of redox balance boosts the antioxidant defenses of stationary phase bacteria. Not only does DksA increase resistance of Salmonella against hydrogen peroxide (H(2)O(2)), but it also promotes fitness of this intracellular pathogen when exposed to oxyradicals produced by the NADPH phagocyte oxidase in an acute model of infection. Given the role of DksA in the adjustment of gene expression in most bacteria undergoing nutritional deprivation, our findings raise the possibility that the control of central metabolic pathways by this regulatory protein maintains redox homeostasis essential for antioxidant defenses in phylogenetically diverse bacterial species.

In eukaryotic cells many cell surface proteins are attached to the membrane via the glycosylphosphatidylinositol (GPI) moiety. In yeast, GPI also plays important roles in the production of mannoprotein in the cell wall. We previously isolated gwt1 mutants and found that GWT1 is required for inositol acylation in the GPI biosynthetic pathway. In this study we isolated a new gwt1 mutant allele, gwt1-10, that shows not only high temperature sensitivity but also low temperature sensitivity. The gwt1-10 cells show impaired acyltransferase activity and attachment of GPI to proteins even at the permissive temperature. We identified TAT2, which encodes a high affinity tryptophan permease, as a multicopy suppressor of cold sensitivity in gwt1-10 cells. The gwt1-10 cells were also defective in the import of tryptophan, and a lack of tryptophan caused low temperature sensitivity. Microscopic observation revealed that Tat2p is not transported to the plasma membrane but is retained in the endoplasmic reticulum in gwt1-10 cells grown under tryptophan-poor conditions. We found that Tat2p was not associated with detergent-resistant membranes (DRMs), which are required for the recruitment of Tat2p to the plasma membrane. A similar result was obtained for Fur4p, a uracil permease localized in the DRMs of the plasma membrane. These results indicate that GPI-anchored proteins are required for the recruitment of membrane proteins Tat2p and Fur4p to the plasma membrane via DRMs, suggesting that some membrane proteins are redistributed in the cell in response to environmental and nutritional conditions due to an association with DRMs that is dependent on GPI-anchored proteins.

Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes. To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E). The strain showed temperature-sensitive growth. Incubation at a nonpermissive temperature (37 degrees C) caused accumulation of cells with nearly 2C DNA content. Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not. Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif. These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication. Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures. We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process.

Malondialdehyde (MDA) is an endogenous genotoxic product of enzymatic and oxygen radical-induced lipid peroxidation whose adducts are known to exist in DNA isolated from healthy human beings. To evaluate the mutagenic potential of MDA in human cells, we reacted MDA with pSP189 shuttle vector DNA and then transfected them into human fibroblasts for replication. MDA induced up to a 15-fold increase in mutation frequency in the supF reporter gene compared with untreated DNA. Sequence analysis revealed that the majority of MDA-induced mutations occurred at GC base pairs. The most frequent mutations were large insertions and deletions, but base pair substitutions were also detected. MDA-induced mutations were completely abolished when the adducted shuttle vector was replicated in cells lacking nucleotide excision repair. MDA induction of large deletions and the apparent requirement for nucleotide excision repair suggested the possible involvement of a DNA interstrand cross-link as a premutagenic lesion. Indeed, MDA formed interstrand cross-links in duplex plasmids and oligonucleotides. Substrates containing the sequence 5'-d(CG) were preferentially cross-linked, consistent with the observation of base pair substitutions in 5'-d(CG) sites in the MDA-induced mutation spectrum. These experiments provide biological and biochemical evidence for the existence of MDA-induced DNA interstrand cross-links that could result from endogenous oxidative stress and likely have potent biological effects.

The serine 202 to phenylalanine substitution within the cytoplasmic interdomain loop of Tet(C) greatly reduces tetracycline resistance and efflux activity (Saraceni-Richards, C. A., and Levy, S. B. (2000) J. Biol. Chem. 275, 6101-6106). Second-site suppressor mutations were identified following hydroxylamine and nitrosoguanidine mutagenesis. Three mutations, L11F in transmembrane 1 (TM1), A213T in the central interdomain loop, and A270V in cytoplasmic loop 8-9, restored a wild type level of resistance and an active efflux activity in Escherichia coli cells bearing the mutant tet(C) gene. The Tet S202F protein with the additional A270V mutation was expressed in amounts comparable with the original mutant, whereas L11F and A213T Tet(C) protein mutants were overexpressed. Introduction of each single mutation into the wild type tet(C) gene by site-directed mutagenesis did not alter tetracycline resistance or efflux activity. These secondary mutations may restore resistance by promoting a conformational change in the protein to accommodate the S202F mutation. The data demonstrate an interaction of the interdomain loop with other distant regions of the protein and support a role of the interdomain loop in mediating tetracycline resistance.

The ATP-dependent molecular chaperone Hsp90 is an essential and abundant stress protein in the eukaryotic cytosol that cooperates with a cohort of cofactors/cochaperones to fulfill its cellular tasks. We have identified Aha1 (activator of Hsp90 ATPase) and its relative Hch1 (high copy Hsp90 suppressor) as binding partners of Hsp90 in Saccharomyces cerevisiae. By using genetic and biochemical approaches, the middle domain of Hsp90 (amino acids 272-617) was found to mediate the interaction with Aha1 and Hch1. Data base searches revealed that homologues of Aha1 are conserved from yeast to man, whereas Hch1 was found to be restricted to lower eukaryotes like S. cerevisiae and Candida albicans. In experiments with purified proteins, Aha1 but not Hch1 stimulated the intrinsic ATPase activity of Hsp90 5-fold. To establish their cellular role further, we deleted the genes encoding Aha1 and Hch1 in S. cerevisiae. In vivo experiments demonstrated that Aha1 and Hch1 contributed to efficient activation of the heterologous Hsp90 client protein v-Src. Moreover, Aha1 and Hch1 became crucial for cell viability under non-optimal growth conditions when Hsp90 levels are limiting. Thus, our results identify a novel type of cofactor involved in the regulation of the molecular chaperone Hsp90.

The fifth and the sixth cytoplasmic regions (C5 and C6) of SecY are important for the SecA-driven preprotein translocation reaction. A cold-sensitive mutation, secY205 (Tyr-429 --> Asp), in C6 impairs the ATP- and precursor-dependent SecA insertion into the membrane. We now identified second site mutations that suppressed the defect. Cis-placement of these mutations proved to suppress mutations at another essential residue (Arg-357) of SecY as well. Thus, they tolerate the otherwise defective SecY alterations in the same molecule. Two alterations (Ile-195 to Ser in TM5 region and Ile-408 to Leu in TM10 region) were found to make the translocation channel more active, because it enabled cells to survive with reduced content of the SecYE complex. These mutations only very weakly suppressed a signal sequence defect of the lambda receptor protein. The mutant SecYEG translocase exhibited higher than normal activity in vitro, being accompanied by striking independence of the proton motive force as well as by stabilization of a bound and active SecA species against urea treatment. These results have been interpreted in terms of balance shifts between channel closing and channel opening alterations in the SecYEG translocase.

The ability of small heat shock proteins (sHSPs) to prevent thermal aggregation of other proteins may require disassembly and reassembly of sHSP oligomers. We investigated the role of changes in sHSP oligomerization by studying a mutant with reduced oligomeric stability. In HSP16.6, the single sHSP in the cyanobacterium Synechocystis sp. PCC 6803, the mutation L66A causes oligomer instability and reduced chaperone activity in vitro. Because thermotolerance of Synechocystis depends on HSP16.6, a phenotype that is enhanced in a deltaClpB1 strain, the effect of mutations can also be assayed in vivo. L66A causes severe defects in thermotolerance, suggesting that oligomeric stability of sHSPs is required for cellular function. This hypothesis was supported by a selection for intragenic suppressors of L66A, which identified mutations that stabilize oligomers of both L66A and wild-type HSP16.6. Analysis of both over- and under-oligomerizing mutants suggests that sHSPs must disassemble before they can release substrates. Furthermore, the suppressor mutations not only restore in vivo activity to L66A, they also ameliorate chaperone defects in vitro, and thus provide the first direct evidence for a chaperone function of an sHSP in cellular thermotolerance.

Elemental manganese is essential for the production of molecular oxygen by cyanobacteria, plants, and algae. In the cyanobacterium Synechocystis sp. PCC 6803, transcription of the mntCAB operon, encoding a high affinity Mn transporter, occurs under Mn starvation (nm Mn) conditions but not in Mn-sufficient (microm Mn) growth medium. Using a strain in which the promoter of this operon directs the transcription of the luxAB reporter genes, we determined that inactivation of the slr0640 gene, which encodes a histidine kinase sensor protein component of a two-component signal transduction system, resulted in constitutive high levels of lux luminescence. Systematic targeted inactivation mutagenesis also identified slr1837 as the gene encoding the corresponding response regulator protein. We have named these two genes manS (manganese-sensor) and manR (manganese-regulator), respectively. A polyhistidine-tagged form of the ManS protein was localized in the Synechocystis 6803 cell membrane. Directed replacement of the conserved catalytic His-205 residue of this protein by Leu abolished its activity, although the mutated protein was present in cyanobacterial membrane. This mutant also showed suboptimal rates of Mn uptake under either Mn-starved or Mn-sufficient growth condition. These data suggest that the ManS/ManR two-component system plays a central role in the homeostasis of manganese in Synechocystis 6803 cells.

The yeast mitochondrial RNA polymerase (RNAP) is a two-subunit enzyme composed of a catalytic core (Rpo41) and a specificity factor (Mtf1) encoded by nuclear genes. Neither subunit on its own interacts with promoter DNA, but the combined holo-RNAP recognizes and selectively initiates from promoters related to the consensus sequence ATATAAGTA. To pursue the question of why Rpo41, which resembles the single polypeptide RNAPs from bacteriophage T7 and T3, requires a separate specificity factor, we analyzed a collection of Mtf1 point mutations that confer an in vivo petite phenotype. These mutant proteins are able to interact with Rpo41 and are capable of nearly wild type levels of initiation in vitro with a consensus promoter-containing template (14 S rRNA). However, the petite phenotype of two mutants can be explained by the fact that they exhibit dramatic transcriptional defects on non-consensus promoters. Y54F is incapable of transcribing the weak tRNA(Cys) promoter, and C192F cannot transcribe either tRNA(Cys) or the variant COX2 promoter from linear DNA templates. Transcription of the tRNA(Cys) promoter by both mutants was significantly corrected by addition of an initiating dinucleotide primer or by supercoiling the DNA template. These results establish the critical role of Mtf1 in promoter recognition and initiation of transcription.

Triplex-forming oligonucleotides (TFOs) bind specifically to duplex DNA and provide a strategy for site-directed modification of genomic DNA. Recently we demonstrated TFO-mediated targeted gene knockout following systemic administration in animals. However, a limitation to this approach is the requirement for a polypurine tract (typically 15-30 base pairs (bp)) in the target DNA to afford high affinity third strand binding, thus restricting the number of sites available for effective targeting. To overcome this limitation, we have investigated the ability of chemically modified TFOs to target a short (10 bp) site in a chromosomal locus in mouse cells and induce site-specific mutations. We report that replacement of the phosphodiester backbone with cationic phosphoramidate linkages, either N,N-diethylethylenediamine or N,N-dimethylaminopropylamine, in a 10-nucleotide, psoralen-conjugated TFO confers substantial increases in binding affinity in vitro and is required to achieve targeted modification of a chromosomal reporter gene in mammalian cells. The triplex-directed, site-specific induction of mutagenesis in the chromosomal target was charge- and modification-dependent, with the activity of N,N-diethylethylenediamine > N,N-dimethylaminopropylamine phosphodiester, resulting in 10-, 6-, and <2-fold induction of target gene mutagenesis, respectively. Similarly, N,N-diethylethylenediamine and N,N-dimethylaminopropylamine TFOs were found to enhance targeting at a 16-bp G:C bp-rich target site in a chromatinized episomal target in monkey COS cells, although this longer site was also targetable by a phosphodiester TFO. These results indicate that replacement of phosphodiester bonds with positively charged N,N-diethylethylenediamine linkages enhances intracellular activity and allows targeting of relatively short polypurine sites, thereby substantially expanding the number of potential triplex target sites in the genome.

Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.

A novel gene, Reprimo, in which induction in cells exposed to X-irradiation is dependent on p53 expression, has been isolated. Ectopic p53 expression results in the induction of its mRNA. Reprimo is a highly glycosylated protein and, when ectopically expressed, it is localized in the cytoplasm and induces G(2) arrest of the cell cycle. In the arrested cells, both Cdc2 activity and nuclear translocation of cyclin B1 are inhibited, suggesting the involvement of Reprimo in the Cdc2.cyclin B1 regulation pathway. Thus, Reprimo may be a new member involved in the regulation of p53-dependent G(2) arrest of the cell cycle.

PTEN/MMAC is a phosphatase that is mutated in multiple human tumors. PTEN/MMAC dephosphorylates 3-phosphorylated phosphatidylinositol phosphates that activate AKT/protein kinase B (PKB) kinase activity. AKT/PKB is implicated in the inhibition of apoptosis, and cell lines and tumors with mutated PTEN/MMAC show increased AKT/PKB kinase activity and resistance to apoptosis. PTEN/MMAC contains a PDZ domain-binding site, and we show here that the phosphatase binds to a PDZ domain of membrane-associated guanylate kinase with inverted orientation (MAGI) 3, a novel inverted membrane-associated guanylate kinase that localizes to epithelial cell tight junctions. Importantly, MAGI3 and PTEN/MMAC cooperate to modulate the kinase activity of AKT/PKB. These data suggest that MAGI3 allows for the juxtaposition of PTEN/MMAC to phospholipid signaling pathways involved with cell survival.

We measured the generation of hydroxyl radical (OH(.)) and oxidative DNA lesions in aerobically grown Escherichia coli cells lacking in both superoxide dismutases (SodA SodB) and repressor of iron uptake (Fur) using electroparamagnetic resonance and gas chromatography-mass spectrometry with a selected-ion monitoring method. A specific signal corresponding to OH(.) generation and an increase in oxidative DNA lesions such as 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine were detected in the strain deficient in sodA sodB fur. We showed that iron metabolism deregulation in fur mutant produced a 2.5-fold iron overload. The sodA sodB fur strain was about 100-fold higher mutability than the wild-type strain. The mutation spectrum in the strain was found to induce GC --> TA and AT --> CG transversions predominantly. The hypermutability of the strain was suppressed by the tonB mutation which reduces iron transport. Thus, excess iron and excess superoxide were responsible for OH(.) generation, oxidative DNA lesion formation, and hypermutability in E. coli.

The eukaryotic 25 S, 18 S, and 5.8 S rRNAs are synthesized as a single transcript with two internal transcribed spacers (ITS1 and ITS2), which are removed by endo- and exoribonucleolytic steps to produce mature rRNA. Genetic selection for suppressors of a polyadenylation defect yielded two cold-sensitive alleles of a gene that we named RRP6 (ribosomal RNA processing). Molecular cloning of RRP6 revealed its homology to a 100-kDa human, nucleolar PM-Scl autoantigen and to Escherichia coli RNase D, a 3'-5' exoribonuclease. Recessive mutations in rrp6 result in the accumulation of a novel 5. 8 S rRNA processing intermediate, called 5.8 S*, which has normal 5' ends, but retains approximately 30 nucleotides of ITS2. Pulse-chase analysis of 5.8 S rRNA processing in an rrp6- strain revealed a precursor-product relationship between 5.8 S* and 5.8 S rRNAs, suggesting that Rrp6p plays a role in the removal of the last 30 nucleotides of ITS2 from 5.8 S precursors. A portion of 5.8 S* rRNA assembles into 60 S ribosomes which form polyribosomes, suggesting that they function in protein synthesis. These findings indicate that Rrp6p plays a role in 5.8 S rRNA 3' end formation, and they identify a functional intermediate in the rRNA processing pathway.

A conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), is located in loop 2/3 and loop 8/9 in the lactose permease, and also in hundreds of evolutionarily related transporters. The importance of conserved residues in loop 8/9 was previously investigated (Pazdernik, N. J., Jessen-Marshall, A. E., and Brooker, R. J. (1997) J. Bacteriol. 179, 735-741). Although this loop was tolerant of many substitutions, a few mutations in the first position of the motif were shown to dramatically decrease lactose transport. In the current study, a mutant at the first position in the motif having very low lactose transport, Leu280, was used as a parental strain to isolate second-site revertants that restore function. A total of 23 independent mutants were sequenced and found to have a second amino acid substitution at several locations (G46C, G46S, F49L, A50T, L212Q, L216Q, S233P, C333G, F354C, G370C, G370S, and G370V). A kinetic analysis revealed that the first-site mutation, Leu280, had a slightly better affinity for lactose compared with the wild-type strain, but its Vmax for lactose transport was over 30-fold lower. The primary effect of the second-site mutations was to increase the Vmax for lactose transport, in some cases, to levels that were near the wild-type value. When comparing this study to second-site mutations obtained from loop 2/3 defective strains, a striking observation was made. Mutations in three regions of the protein, codons 45-50, 234-241, and 366-370, were able to restore functionality to both loop 2/3 and loop 8/9 defects. These results are discussed within the context of a C1/C2 alternating conformation model in which lactose translocation occurs by a conformational change at the interface between the two halves of the protein.

The GTPase Ran/TC4 and the 14-kDa protein nuclear transport factor 2 (NTF2) are two of the cytosolic factors that mediate nuclear protein import in vertebrates. Previous biochemical studies have shown that NTF2 binds directly to the GDP-bound form of Ran/TC4 and to proteins of the nuclear pore complex that contain phenylalanine-glycine repeats. In the present study we have used molecular genetic approaches to study the Saccharomyces cerevisiae homologue of NTF2. The scNTF2 gene encodes a protein that is 44% identical to the human protein. We found that deletion of the scNTF2 gene is lethal and that repression of NTF2p expression by a regulatable promoter results in gross structural distortions of the nuclear envelope. In a screen for high copy number suppressors of a scNTF2 deletion, the only gene we isolated other than scNTF2 itself was GSP1, the S. cerevisiae homologue of Ran/TC4. Furthermore, we found that high levels of Ran/TC4 can relieve the requirement for NTF2 in a mammalian-permeabilized cell assay for nuclear protein import. These data suggest that certain of the nuclear protein import functions of NTF2 and Ran/TC4 are closely linked and that NTF2 may serve to modulate a transport step involving Ran/TC4.

Overexpression of the Escherichia coli msbB gene on high copy plasmids suppresses the temperature-sensitive growth associated with mutations in the htrB gene. htrB encodes the lauroyl transferase of lipid A biosynthesis that acylates the intermediate (Kdo)2-lipid IVA (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem. 265, 15410-15417). Since msbB displays 27.5% identity and 42.2% similarity to htrB, we explored the possibility that msbB encodes a related acyltransferase. In contrast to htrB, extracts of strains with insertion mutations in msbB are not defective in transferring laurate from lauroyl acyl carrier protein to (Kdo)2-lipid IVA. However, extracts of msbB mutants do not efficiently acylate the product formed by HtrB, designated (Kdo)2-(lauroyl)-lipid IVA. Extracts of strains harboring msbB+ bearing plasmids acylate (Kdo)2-(lauroyl)-lipid IVA very rapidly compared with wild type. We solubilized and partially purified MsbB from an overproducing strain, lacking HtrB. MsbB transfers myristate or laurate, activated on ACP, to (Kdo)2-(lauroyl)-lipid IVA. Decanoyl, palmitoyl, palmitoleoyl, and (R)-3-hydroxymyristoyl-ACP are poor acyl donors. MsbB acylates (Kdo)2-(lauroyl)-lipid IVA about 100 times faster than (Kdo)2-lipid IVA. The slow, but measurable, rate whereby MsbB acts on (Kdo)2-lipid IVA may explain why overexpression of MsbB suppresses the temperature-sensitive phenotype of htrB mutations. Presumably, the acyloxyacyl group generated by excess MsbB substitutes for the one normally formed by HtrB.

C129/U1 is a respiratory defective mutant of Saccharomyces cerevisiae arrested in cytochrome oxidase assembly due to a mutation in COX17, a nuclear gene encoding a low molecular weight cytoplasmic protein proposed to function in mitochondrial copper recruitment. In the present study we show that the respiratory defect of C129/U1 is rescuable by two multicopy suppressors, SCO1 and SCO2. SCO1 was earlier reported to code for a mitochondrial inner membrane protein with an essential function in cytochrome oxidase assembly (Buchwald, P., Krummeck, G., and Rodel, G. (1991) Mol. Gen. Genet. 229, 413-420). SCO2 is a homologue of SCO1, whose product is also localized in the mitochondrial membrane but is not required for respiration. SCO1 also suppresses a cox17 null mutant, indicating that overexpression of Sco1p can compensate for the absence of Cox17p. In contrast, neither copper, COX17 on a multicopy plasmid, or a combination of the two is able to restore respiration in sco1 mutants. Rescue of cox17 mutants by Sco1p suggests that this mitochondrial protein plays a role either in mitochondrial copper transport or insertion of copper into the active site of cytochrome oxidase. Although SCO2 can also partially restore respiratory growth in the cox17 null mutant, rescue in this case requires addition of copper to the growth medium. SCO2 does not suppress a sco1 null mutant, although it is able to partially rescue a sco1 point mutant. We interpret the ability of SCO2 to restore respiration in cox17, but not in sco1 mutants, to indicate that Sco1p and Sco2p have overlapping but not identical functions.

A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligand-receptor interactions in the prototypic seven-transmembrane neurokinin-2 receptor (NK2) using this fluorescence-based approach is presented. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants containing an unique 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dap) residue at either site 103, in the first extracellular loop, or 248, in the third cytoplasmic loop, were functionally active. The fluorescent NK2 mutants were investigated by microspectrofluorimetry in a native membrane environment. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calculated by the theory of Förster, permit to fix the ligand in space and define the structure of the receptor in a molecular model for NK2 ligand-receptor interactions. Our data are the first report of the incorporation of a fluorescent unnatural amino acid into a membrane protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of experimental distances between a G protein-coupled receptor and its ligand by FRET. The method presented here can be generally applied to the analysis of spatial relationships in integral membrane proteins such as receptors or channels.

Expression of the adenine nucleotide translocator 2 (ANT2) gene is growth regulated. We report a feature of the ANT2 promoter that involves a novel regulatory function for the Sp1 transfactor. We show that expression from the ANT2 proximal promoter is modulated through three Sp1 elements, two of which activate and one of which partially inhibits transcription. The inhibitor site, box C, is juxtaposed to transcription start (nucleotides -7 to -2). Sp1 bound to box C decreases transcription initiation. This was demonstrated by introducing mutations in box C which (a) increased chloramphenicol acetyltransferase expression in the transient transfection assay and (b) inhibited binding of both purified Sp1 and Sp1 in crude nuclear extracts. The activating elements (A and B boxes) are located at adjacent sites in the distal region of the proximal promoter. Mutation of either box inhibits transfection by 90%, indicating that they act in a synergistic manner. Supershift experiments with crude nuclear extracts showed that only Sp1 was bound to the three GC boxes. The finding that Sp1 acts as an activator/inhibitor within the same promoter region was verified in NIH3T3, HeLa, JEG3, and COS-1, indicating that this dual effect of Sp1 is widely preserved. These data suggest a unique role for Sp1 and raise the possibility that growth activation of the ANT2 gene is regulated by the interaction of Sp1 on the A, B, and C boxes.

A screen was designed to identify temperature-sensitive mutants of Saccharomyces cerevisiae, whose transcription of both ribosomal RNA and ribosomal protein genes is repressed at the nonpermissive temperature. The gene from one such mutant was cloned by complementation. The gene encodes a predicted product that is nearly 65% identical to the human GTPase, Rab6, and is likely to be identical to the yeast gene YPT6. It is essential for growth only at elevated temperatures. The mutant strain is partially defective in the maturation of the vacuolar protein carboxypeptidase Y, as well as in the secretion of invertase, which accumulates as a core-glycosylated form characteristic of the endoplasmic reticulum or the cis-Golgi, suggesting that Ypt6p is involved in an early step of the secretory pathway, earlier than that reported for the mammalian Rab6. The mutant protein, a truncation at codon 64 of 215, has a stronger phenotype than the null allele of YPT6. Four other mutants selected for defective ribosome synthesis at the nonpermissive temperature were also found to have defects in carboxypeptidase Y maturation, giving emphasis to our previous finding that a functional secretory pathway is essential for continued ribosome synthesis. Cloning of extragenic suppressors of the ts allele of YPT6 has revealed two additional proteins that influence the secretory pathway: Ssd1p, a suppressor of many mutations, and Imh1p, which bears some homology to the C-terminal portion of the cytoskeletal proteins integrin and myosin.

A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), is found in a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. This motif is located within the cytoplasmic side of transmembrane domain 2 (TM-2) and extends through the hydrophilic loop that connects transmembrane domains 2 and 3. The motif is repeated again in the second half of the protein. In a previous study concerning the loop 2/3 motif (Jessen-Marshall, A. E., Paul, N. J., and Brooker, R. J. (1995) J. Biol. Chem. 270, 16251-16257), it was shown that the conserved aspartate at the fifth position in the motif is critical for transport activity since a variety of site-directed mutations were found to greatly diminish the rate of transport. In the current study, two of these mutations, in which the conserved aspartate was changed to threonine or serine, were used as parental strains to isolate second site suppressor mutations that restore transport function. A total of 10 different second site mutations were identified among a screen of 19 independent mutants. One of the suppressors was found within loop 1/2 in which Thr-45 was changed to arginine. Since the conserved aspartate and position 45 are at opposite ends of TM-2, these results suggest that the role of the conserved aspartate residue in loop 2/3 is to influence the topology of TM-2. Surprisingly, the majority of suppressor mutations were found in the second half of the permease. All of these are expected to alter helix topology in either of two ways. Some of the mutations involved residues within transmembrane segments 7 and 11 that produced substantial changes in side chain volume: TM-7 (Cys-234-->Trp or Phe, Gln-241-->Leu, and Phe-247-->Val) and TM-11 (Ser-366-->Phe). Alternatively, other mutations were highly disruptive substitutions at the ends of transmembrane segments or within hydrophilic loops (Gly-257-->Asp, Val-367-->Glu, Ala-369-->Pro, and a 5-codon insertion into loop 11/12). It is hypothesized that the effects of these suppressor mutations are to alter the helical topologies in the second half of the protein to facilitate a better interaction with the first half. Overall, these results are consistent with a transport model in which TM-2 acts as an important interface between the two halves of the lactose permease. According to our tertiary model, this interaction occurs between TM-2 and TM-11.

Oligonucleotides can bind as third strands of DNA in a sequence-specific manner to form triple helices. Psoralen-conjugated, triplex-forming oligonucleotides (TFOs) have been used for the site-specific modification of DNA to inhibit transcription and to target mutations to selected genes. Such strategies, however, must take into account the ability of the cell to repair the triplex-directed lesion. We report experiments showing that the pattern of mutations produced by triplex-targeted psoralen adducts in an SV40 shuttle vector in monkey COS cells can be influenced by the associated third strand. Mutations induced by psoralen adducts in the context of a TFO of length 10 were the same as those generated by isolated adducts but were found to be different from those generated in the presence of a TFO of length 30 at the same target site. In complementary experiments, HeLa whole cell extracts were used to directly assess repair of the TFO-directed psoralen adducts in vitro. Excision of the damaged DNA was inhibited in the context of the 30-mer TFO, but not the 10-mer. These results suggest that an extended triple helix of length 30, which exceeds the typical size of the nucleotide excision repair patch in mammalian cells, can alter repair of an associated psoralen adduct. We present a model correlating these results and proposing that the incision steps in nucleotide excision repair in mammalian cells can be blocked by the presence of a third strand of sufficient length and binding affinity, thereby changing the pattern of mutations. These results may have implications for the use of triplex-forming oligonucleotides for genetic manipulation, and they may lead to the use of such oligonucleotides as tools to probe DNA repair pathways.

Analysis of a series of human beta-myosin heavy chain (MHC) constructs with progressive deletions in the 5'-flanking region has localized a strong positive element at positions -298/277 with a repressor region located immediately upstream at -332/-300 (Flink, I. L., Edwards, J. G., Bahl, J. J., Liew, C.-C., Sole, M., and Morkin, E. (1992) J. Biol. Chem. 267, 9917-9924). A 49-base pair restriction fragment containing the suppressor element was used to screen a cardiac expression library. The 0.65-kilobase pair cDNA identified by this procedure was similar in sequence, except for the absence of a 21-base pair region encoding seven amino acids, to cellular nucleic acid-binding protein (CNBP), a 19-kDa zinc finger DNA-binding protein isolated earlier from liver, which may be involved in negative regulation of cholesterol biosynthesis (Rajavashisth, T. B., Taylor, A. K., Andalibi, A., Svenson, K. L., and Lusis, A. J. (1989) Science 245, 640-643). An additional clone identical to the one originally found in liver, referred to as CNBP alpha, was isolated from the cardiac library by hybridization screening. Gel mobility shift analysis indicated that CNBP alpha and CNBP beta isoforms preferentially interact with single-stranded DNA corresponding to the proximal and distal regions of the suppressor. When cotransfected with a beta-MHC reporter construct, CNBP alpha repressed activity in a dosage-dependent manner, whereas repression was not observed with the shorter construct (CNBP beta). Cotransfection of a combination of CNBP alpha and CNBP beta repressed reporter activity to an extent similar to cotransfection with CNBP alpha alone, suggesting that CNBP beta is not translationally active under these conditions. The results of RNase protection assays and genomic sequencing indicated that the alpha and beta isoforms are formed by alternative use of 5' donor sites within a single exon. These results suggest that CNBP isoforms may modulate the activity of the beta-MHC gene by interaction with a repressor region.

The peripherin gene, which encodes a neuronal-specific intermediate filament protein, is transcriptionally induced with a late time course when nerve growth factor stimulates PC12 cells to differentiate into neurons. We have defined a negative regulatory element (NRE) that has a functional role in repressing peripherin expression in undifferentiate and nonneuronal cells. Nerve growth factor-induced derepression of peripherin gene expression is associated with alterations in proteins binding to a GC-rich DNA sequence in the NRE as detected by the DNA electrophoretic mobility shift assay (EMSA). We have utilized DNA affinity chromatography to purify from rat liver a 33-kDa DNA-binding protein that specifically recognizes the NRE. Microsequencing reveals identity with NF1-L, a member of the CTF/NF-1 transcription factor family. This protein forms a single complex when incubated with the NRE probe using EMSA analysis. The more slowly migrating complexes characteristic of crude undifferentiated PC12 cell extract are reconstituted by mixing the purified protein with the flow-through from the DNA affinity column, thereby demonstrating that protein-protein interactions are involved in complex formation. Supershift experiments incubating anti-CTF-1 antibody with undifferentiated PC12 cell extract prior to EMSA analysis confirm that NF1-L, or a closely related family member, is the DNA-binding protein component of the multiprotein complex at the NRE.

A mutant form of SecY, SecY-d1, was previously suggested to sequester a component(s) of the protein translocator complex. Its synthesis from a plasmid leads to interference with protein export in Escherichia coli. SecE is a target of this sequestration, and its overproduction cancels the export interference. We now report that overexpression of another gene, termed syd, also suppresses secY-d1. The nucleotide sequence of syd predicted that it encodes a protein of 181 amino acid residues, which has been identified by overproduction, purification, and determination of the amino-terminal sequence. Cell fractionation experiments suggested that Syd is loosely associated with the cytoplasmic surface of the cytoplasmic membrane. SecY may be involved in the membrane association of Syd since the association is saturable, the extent of which depends on the overproduction of SecY. SecY is rapidly degraded in vivo unless its primary partner, SecE, is sufficiently available. Overproduction of Syd was found to stabilize oversynthesized SecY. However, Syd cannot stabilize the SecY-d1 form of SecY. Thus, in the presence of both secY+ and secY-d1, Syd increases the effective SecY+/SecY-d1 ratio in the cell and cancels the dominant interference by the latter. We also found that overproduction of Syd dramatically inhibits protein export in the secY24 mutant cell in which SecY-SecE interaction has been weakened. These results indicate that Syd, especially when it is overproduced, has abilities to interact with SecY. Possible significance of such interactions is discussed in conjunction with the apparent lack of phenotypic consequences of genetic disruption of syd.

Two rpoN-linked delta Tn10-kan insertions suppress the conditionally lethal erats allele. One truncates rpoN while the second disrupts another gene (ptsN) in the rpoN operon and does not affect classical nitrogen regulation. Neither alter expression of era indicating that suppression is post-translational. Plasmid clones of ptsN prevent suppression by either disruption mutation indicating that this gene is important for lethality caused by erats. rpoN and six neighboring genes were sequenced and compared with sequences in the database. Two of these genes encode proteins homologous to Enzyme IIAFru and HPr of the phosphoenolpyruvate:sugar phosphotransferase system. We designate these proteins IIANtr (ptsN) and NPr (npr). Purified IIANtr and NPr exchange phosphate appropriately with Enzyme I, HPr, and Enzyme IIA proteins of the phosphoenolpyruvate: sugar phosphotransferase system. Several sugars and tricarboxylic acid cycle intermediates inhibited growth of the ptsN disruption mutant on medium containing an amino acid or nucleoside base as a combined source of nitrogen, carbon, and energy. This growth inhibition was relieved by supplying the ptsN gene or ammonium salts but was not aleviated by altering levels of exogenously supplied cAMP. These results support our previous proposal of a novel mechanism linking carbon and nitrogen assimilation and relates IIANtr to the unknown process regulated by the essential GTPase Era.

We have characterized several mutants that contain alterations in the hydrophilic domain (N region) of the pseudorabies virus glycoprotein gC signal sequence. In general, our results agree with previous findings and indicate that basic residues in the N region are not essential for efficient export of gC in infected cells. While reducing the N region to a net neutral charge led to a slight impairment of membrane translocation, a substantial gC export defect was not observed until a net negative charge was introduced. However, there was one exception to this pattern. The substitution of a leucine for an arginine at the carboxyl terminus of the N region led to a considerable export defect despite maintaining a net positive charge. As a consequence of the substitution, the mutant signal sequence was 1.5 times more hydrophobic than wild type, but we found that the defect could be largely corrected if an additional alteration that lessened the overall hydrophobicity of the gC signal sequence was incorporated. We suggest that an upper limit of hydrophobicity may exist for eukaryotic signal sequences; exceeding this value could lead to an export defect.

We report the characterization of a mutation affecting tau 138, the largest subunit of yeast transcription factor IIIC (TFIIIC). A previously described thermosensitive mutation (tsv115), tightly linked to the centromere of chromosome I (Harris, S.D., and Pringle, J.R. (1991) Genetics 127, 279-285) is shown to lie in the TFC3 gene which encodes tau 138. The tau 138 subunit carrying this mutation bears a single substitution of Glu for Gly at position 349 (G349E). In extracts from mutant cells, both the level of TFIIIC and its affinity for tDNA were found to be reduced. The tDNA binding activity of mutant TFIIIC protein was very sensitive to mild heat treatments, and TFIIIC-DNA interaction was inhibited at moderate salt concentrations, as evidenced by gel shift assays. In addition, the tsv115 mutation affected 5 S RNA synthesis in vitro, suggesting that the tau 138 subunit also plays a role in recognition of the TFIIIA-5 S DNA complex. Multicopy suppressors of the TFIIIC defect were sought to reveal components participating in TFIIIC function. One class of suppressors encodes known components of the transcription machinery: two TFIIIC subunits, tau 95 and tau 131, the 70-kDa subunit of TFIIIB, TBP, and a shared subunit of RNA polymerase (pol) I, II, and III, ABC10 alpha; it also includes genes potentially related to pol III function, such as SRP40 which also suppresses a mutation in a subunit shared by RNA polymerases I and III. A second class of suppressors is not involved in transcription but alleviates the main physiological defects of mutant cells. It includes RPR1 and NOP1, required for the maturation of pre-tRNA and pre-rRNA, respectively.

We have explored the role of the distal switch II region of the yeast RAS2 protein in determining the response to the nucleotide exchange factor CDC25. We first constructed yeast tester strains in which the deletion of the chromosomal CDC25, RAS1, and RAS2 genes, in combination with the chromosomal suppressor CRI4, resulted in detectable phenotypes in vivo and in vitro. Phenotypes included impaired growth at 37 degrees C, defective glucose-induced cyclic AMP signaling, and low adenylyl cyclase activity of membrane preparations. Tester strains were subsequently used for the reintroduction of various combinations of wild-type and mutated RAS2 and CDC25 genes by genetic techniques, as well as for in vitro reconstitution assays with the corresponding proteins. CDC25 restored both growth and glucose-induced cyclic AMP signaling in the presence, but not in the absence of wild-type RAS2. A gene encoding a RAS2 protein with a mutationally altered switch II region was expressed but was ineffective in reintegrating exchange factor-dependent responses in vivo. Wild-type, but not mutagenically altered, RAS2 proteins were stimulated by exchange factors in vitro. We conclude that the conserved distal switch II region is required for CDC25-dependent activation of RAS.

The Q42E mutation in the polar loop of subunit c of the Escherichia coli F1F0 ATP synthase leads to an uncoupling of H+ translocation through F0 and ATP synthesis/hydrolysis in F1. We have isolated four second-site suppressor mutants in which the coupling defect is corrected. Substitutions for Glu31 in F1 subunit epsilon were found in each suppressor mutant, where the substitutions were E31G, E31V, and E31K (the last being found twice). The different substitutions vary in effectiveness in restoring wild type growth properties in the order epsilon E31G > epsilon E31V > epsilon E31K. Biochemical properties of epsilon E31G/cQ42E and epsilon E31K/cQ42E membranes were compared. In epsilon E31G/cQ42E mutant membranes, ATP-driven H+ translocation by F1F0 and the binding and coupling of F1 to F0 showed a striking pH dependence. Near normal function was observed at pH 7.0, but function was lost at pH 7.8. The function of epsilon E31K/cQ42E membranes was much less affected by changes in pH. Relative to epsilon E31G/cQ42E membranes, the ATP-driven H+ transport function of epsilon E31K/cQ42E membranes was approximately the same at pH 7.5, greater at pH 7.8, and less at pH 7.0. The differences between mutants could be explained if cGlu42 ionized at pH 7.8 with loss of function in epsilon E31G/cQ42E membrane and a similar ionization were compensated for by the positively charged Lys in the epsilon E31K/cQ42E membrane.

The acceptor helix of histidine tRNAs in Escherichia coli is capped by a unique base pair in which the cytosine at the discriminator position is paired with an extra guanosine at -1. In previous in vitro studies, the presence of the G-1:C73 base pair was found to be required to obtain both optimal histidylation by histidyl-tRNA synthetase and accurate 5' processing by RNase P. We investigated the role of G-1:C73 in histidine tRNA identity and found that nucleotide substitutions conferred mischarging by other amino acids in a pattern that correlated with the discriminator base and not with the extra nucleotide at -1. As shown by primer extension experiments, the relatively minor role of the -1 nucleotide in vivo could be attributed to altered RNase P processing. These studies show that interactions of tRNAs in vivo both with RNase P during tRNA biosynthesis and with the pool of aminoacyl-tRNA synthetases can modulate the effects of substitutions at recognition nucleotides, eliciting changes in transfer RNA identity.