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Degradation of p53, not telomerase activation, by E6 is required for bypass of crisis and immortalization by human papillomavirus type 16 E6/E7
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Bypass of two arrest points is essential in the process of cellular immortalization, one of the components of the transformation process. Expression of human papillomavirus type 16 E6 and E7 together can escape both senescence and crisis, processes which normally limit the proliferative capacity of primary human keratinocytes. Crisis is thought to be mediated by telomere shortening. Because E6 stimulates telomerase activity and exogenous expression of the TERT gene with E7 can immortalize keratinocytes, this function is thought to be important for E6 to cooperate with E7 to bypass crisis. However, it has also been reported that E6 dissociates increased telomerase activity from maintenance of telomere length and that a dominant-negative p53 molecule can substitute for E6 in cooperative immortalization of keratinocytes with E7. Thus, to determine which functions of E6 are required to allow bypass of crisis and immortalization of keratinocytes with E7, immortalization assays were performed using specific mutants of E6, in tandem with E7. In these experiments, every clone expressing an E6 mutant capable of degrading p53 was able to bypass crisis and immortalize, regardless of telomerase induction. All clones containing E6 mutants incapable of degrading p53 died at crisis. These results suggest that the ability of E6 to induce degradation of p53 compensates for continued telomere shortening in E6/E7 cells and demonstrate that degradation of p53 is required for immortalization by E6/E7, while increased telomerase activity is dispensable. 相似文献
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The major subunit of Escherichia coli type 1 fimbriae is not required for D-mannose-specific adhesion 总被引:8,自引:0,他引:8
Type 1 fimbriae are surface organelles on Escherichia coli, which mediate specific binding to D-mannose-containing structures. These fimbriae are heteropolymers composed of a major building element, the FimA protein, and small amounts of the FimF, FimG and FimH proteins. The FimH protein is uniquely responsible for the D-mannose receptor binding. In this work data are presented which indicate that the major subunit of type 1 fimbriae is dispensable for D-mannose-specific binding. A recombinant strain was studied which harboured an insertional deletion in the fimA gene, and was thereby unable to produce type 1 fimbriae; however, it was still able to express a D-mannose-binding phenotype. However, the deletion resulted in a 25-fold reduction of the adhesive potential, as measured by binding to D-mannose-coated Sepharose beads. Serological and specific receptor binding evidence is presented that suggests that the FimH adhesion is capable of being exposed on the bacterial surface without being an integral part of the fimbriae. 相似文献
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The Escherichia coli RNase E is an essential endoribonuclease involved in processing and/or degradation of rRNAs, tRNAs, and non-coding small RNAs as well as many mRNAs. It is known that RNase E activity is somehow regulated by an RNA-binding protein Hfq, at least in some cases. We searched for proteins that showed changes in expression in both hfq::cat and rne-1 mutant cells as compared with the wild type, and found that a protein band of 49-kDa decreased in these mutant cells at 42 degrees C, the restrictive temperature for rne-1. N-terminal amino acid sequencing identified it as a mixture of GadA and GadB, two isozymes of glutamate decarboxylase involved in glutamate-dependent acid resistance. The rne-1 mutant as well as the hfq mutant showed decreased survival under acidic conditions (pH 2.5). Hfq is known to regulate the expression of GadA/B in RpoS- and GadY small RNA-dependent ways. We examined the expression of these two regulators in rne-1 mutant cells. In the mutant cells, the induction of GadY was defective at 42 degrees C, but the expression of RpoS was normal. These results indicate that RNase E is required for induction of the glutamate-dependent acid resistance system in a RpoS-independent manner. 相似文献
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lep operon proximal gene is not required for growth or secretion by Escherichia coli. 总被引:1,自引:0,他引:1
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Leader peptidase is an essential enzyme of Escherichia coli and is required for protein export. The structural gene for leader peptidase (lep) is separated from its promoter by an upstream gene of unknown function (lepA). The gene lepA was shown by the use of minicell analysis and overproduction to encode a protein of 74,000 daltons. To determine whether this 74,000-dalton protein functions in protein export, a mutant of E. coli H560 was constructed which has a 1.5-kilobase-pair deletion in the lepA gene. The lepA deletion mutant had no apparent defect for growth or protein export, indicating that lepA is nonessential and that the two cotranscribed genes lepA and lep probably have unrelated functions. 相似文献
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Adenylate cyclase is required for chemotaxis to phosphotransferase system sugars by Escherichia coli. 总被引:8,自引:5,他引:3
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We report that in Escherichia coli, chemotaxis to sugars transported by the phosphotransferase system is mediated by adenylate cyclase, the nucleotide cyclase linked to the phosphotransferase system. We conclude that adenylate cyclase is required in this chemotaxis pathway because mutations in the cyclase gene (cya) eliminate or impair the response to phosphotransferase system sugars, even though other components of the phosphotransferase system known to be required for the detection of these sugars are relatively unaffected by such mutations. Moreover, merely supplying the mutant bacteria with the products of this enzyme, cyclic AMP and cyclic GMP, does not restore the chemotactic response. Because a residual chemotactic response is observed in certain strains with residual cyclic GMP synthesis but no cyclic AMP synthesis, it appears that the guanylate cyclase activity rather than the adenylate cyclase activity of the enzyme may be required for chemotaxis to sugars transported by the phosphotransferase system. Mutations in the cyclic nucleotide phosphodiesterase gene, which increase the level of both cyclic AMP and cyclic GMP, also reduce chemotaxis to these sugars. Therefore, it appears that control of the level of a cyclic nucleotide is critical for the chemotactic response to phosphotransferase system sugars. 相似文献
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Adenovirus type 12 E1B 19-kilodalton protein is not required for oncogenic transformation in rats. 总被引:7,自引:7,他引:0
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The adenovirus type 12 mutants in700 and pm700 carry site-specific mutations within the reading frame encoding the E1B 19-kilodalton protein (19K protein) which prevent the production of the intact 19K protein. In cultures of human A549 cells, these mutants grow just as well as the wild-type virus does, but they display a large-plaque (lp), cytocidal (cyt) phenotype. DNA in these infected cells is not degraded, but at late times in human KB cells infected by the mutants, the mutants display a DNA degradation (deg) phenotype. The transformation phenotype of these mutants is also host range. Although the mutants are defective for transformation of the 3Y1 rat cell line, they transform rat and mouse primary kidney cells in vitro at wild-type efficiency and are capable of inducing tumors in rats. These results support the view that the type 12 E1B 19K protein is not obligatory for oncogenic transformation. 相似文献
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A strain of Escherichia coli lacking RNAase III and containing thermolabile RNAase E and RNAase P was labeled with 32Pi at a non-permissive temperature. RNA molecules were separated by two-dimensional polyacrylamide gel electrophoresis. Most of the small RNA species were isolated and analyzed for the presence of 5′ nucleoside triphosphates. In 16 of the 22 species analyzed a significant number of the individual molecules contained 5′ di or triphosphates. We conclude, therefore, that very little endonucleolytic RNA processing occurs in the absence of the three RNA processing enzymes RNAase III, RNAase E and RNAase P. 相似文献
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Homology is not required for recombination mediated by DNA gyrase of Escherichia coli 总被引:10,自引:0,他引:10
Summary We have previously shown that DNA gyrase of Escherichia coli can promote recombination between heterologous DNAs in a cell-free system (Ikeda et al. 1982). In the present paper, we have studied the nucleotide sequences of several recombination junctions of -pBR322 recombinants and found that there were not more than three-basepair homologies between the parental DNAs in six combinations of the and pBR322 recombination sites. Based on this and previous results, we concluded that homology was not required for the DNA gyrase-mediated recombination. Furthermore, the structures of the recombinant DNAs we have analyzed suggest the occurrence of multiple crossovers in our in vitro system. 相似文献
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6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well. 相似文献
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The PDZ ligand domain of the human papillomavirus type 16 E6 protein is required for E6's induction of epithelial hyperplasia in vivo 总被引:11,自引:0,他引:11
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Human papillomaviruses (HPVs) are the causative agent of warts. Infections with high-risk HPVs are associated with anogenital and head and neck cancers. One of the viral genes responsible for HPV's oncogenic activity is E6. Mice expressing the HPV-16 E6 protein in their epidermis (K14E6(WT)) develop epithelial hyperplasia and squamous carcinomas. Numerous cellular proteins interact with E6, some of which can be grouped based on common amino acid motifs in their E6-binding domains. One such group, the PDZ partners, including hDLG, hSCRIBBLE, MUPP1, and MAGI, bind to the carboxy-terminal four amino acids of E6 through their PDZ domains. E6's interaction with the PDZ partners leads to their degradation. Additionally, E6's binding to PDZ proteins has been correlated with its ability to transform baby rat kidney cells in tissue culture and to confer tumorigenicity onto cells in xenograft experiments. To address whether the ability of E6 to bind PDZ domain partners is necessary for E6 to confer epithelial hyperproliferation in vivo, we generated transgenic mice that express in stratified squamous epithelia a mutant of E6 lacking the last six amino acids at its carboxyl terminus, E6(Delta 146-151), from the human keratin 14 (K14) promoter. The K14E6(Delta 146-151) mice exhibit a radiation response similar to that of the K14E6(WT) mice, demonstrating that this protein, as predicted, retains an ability to inactivate p53. However, the K14E6(Delta 146-151) mice fail to display epithelial hyperplasia. These results indicate that an interaction of E6 with PDZ partners is necessary for its induction of epithelial hyperplasia. 相似文献
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The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity
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Anne Chapman-Smith Terrence D. Mulhern Fiona Whelan John E. Cronan Jr. John C. Wallace 《Protein science : a publication of the Protein Society》2001,10(12):2608-2617
Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5'-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain. 相似文献
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Olga Musharova Vasily Sitnik Marnix Vlot Ekaterina Savitskaya Kirill A. Datsenko Andrey Krivoy Ivan Fedorov Ekaterina Semenova Stan J. J. Brouns Konstantin Severinov 《Molecular microbiology》2019,111(6):1558-1570
CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR‐Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR‐Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I‐E CRISPR‐Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition. 相似文献
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Escherichia coli DnaA protein initiates DNA replication from the chromosomal origin, oriC, and regulates the frequency of this process. Structure-function studies indicate that the replication initiator comprises four domains. Based on the structural similarity of Aquifex aeolicus DnaA to other AAA+ proteins that are oligomeric, it was proposed that Domain III functions in oligomerization at oriC (Erzberger, J. P., Pirruccello, M. M., and Berger, J. M. (2002) EMBO J. 21, 4763-4773). Because the Box VII motif within Domain III is conserved among DnaA homologues and may function in oligomerization, we substituted conserved Box VII amino acids of E. coli DnaA with alanine by site-directed mutagenesis to examine the role of this motif. All mutant proteins are inactive in initiation from oriC in vivo and in vitro, but they support RK2 plasmid DNA replication in vivo. Thus, RK2 requires only a subset of DnaA functions for plasmid DNA replication. Biochemical studies on a mutant DnaA carrying an alanine substitution at arginine 281 (R281A) in Box VII show that it is inactive in in vitro replication of an oriC plasmid, but this defect is not from the failure to bind to ATP, DnaB in the DnaB-DnaC complex, or oriC. Because the mutant DnaA is also active in the strand opening of oriC, whereas DnaB fails to bind to this unwound region, the open structure is insufficient by itself to load DnaB helicase. Our results show that the mutant fails to form a stable oligomeric DnaA-oriC complex, which is required for the loading of DnaB. 相似文献
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Pre-steady-state chemical quenched-flow techniques were used to study DNA unwinding catalyzed by Escherichia coli UvrD helicase (helicase II), a member of the SF1 helicase superfamily. Single turnover experiments, with respect to unwinding of a DNA oligonucleotide, were used to examine the DNA substrate and UvrD concentration requirements for rapid DNA unwinding by pre-bound UvrD helicase. In excess UvrD at low DNA concentrations (1 nM), the bulk of the DNA is unwound rapidly by pre-bound UvrD complexes upon addition of ATP, but with time-courses that display a distinct lag phase for formation of fully unwound DNA, indicating that unwinding occurs in discrete steps, with a "step size" of four to five base-pairs as previously reported. Optimum unwinding by pre-bound UvrD-DNA complexes requires a 3' single-stranded (ss) DNA tail of 36-40 nt, whereas productive complexes do not form readily on DNA with 3'-tail lengths =16 nt. A 5'-ssDNA tail is neither sufficient nor does it stimulate unwinding, even in the presence of a 3'-ssDNA tail. Nitrocellulose filter binding studies show that UvrD binding affinity also increases with increasing 3'-ssDNA tail length, showing apparent positive cooperativity for binding to DNA with a 40 nt 3'-ssDNA tail. Single turnover DNA unwinding experiments performed at higher DNA concentrations (50 nM) show a sigmoidal dependence of the extent of unwinding on the pre-incubated [UvrD], also indicating cooperativity. These results indicate that the form of the UvrD helicase with optimal helicase activity is oligomeric with at least two sites for binding the DNA substrate, where one site contacts regions of the 3'-ssDNA tail that are distal from the single-stranded/double-stranded DNA junction. 相似文献
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Ando T Tanaka T Hori Y Sakai E Kikuchi Y 《Bioscience, biotechnology, and biochemistry》2001,65(12):2798-2801
Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNA(Ala), tRNA(His), and tRNA(iMet)) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool detect destablized tRNA molecules from any species. 相似文献
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An arginine-faced amphipathic alpha helix is required for adenovirus type 5 e4orf6 protein function
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A region in the carboxy terminus of the protein encoded by open reading frame 6 in early region 4 (E4orf6) of adenovirus type 5 was determined to be required for directing nuclear localization of the E1B 55-kDa protein and for efficient virus replication. A peptide encompassing this region, corresponding to amino acids 239 through 255 of the E4orf6 protein, was analyzed by circular dichroism spectroscopy. The peptide showed evidence of self-interaction and displayed the characteristic spectra of an amphipathic alpha helix in the helix-stabilizing solvent trifluoroethanol. Disrupting the integrity of this alpha helix in the E4orf6 protein by proline substitutions or by removing amino acids 241 through 250 abolished its ability to direct the E1B 55-kDa protein to the nucleus when both proteins were transiently expressed in HeLa cells. Expression of E4orf6 variants that failed to direct nuclear localization of the E1B 55-kDa protein failed to enhance replication of the E4 mutant virus, dl1014, whereas expression of the wild-type E4orf6 protein restored growth of dl1014 to near-wild-type levels. These results suggest that the E4orf6 protein contains an arginine-faced, amphipathic alpha helix that is critical for a functional interaction with the E1B 55-kDa protein in the cell and for the function of the E4orf6 protein during a lytic infection. 相似文献