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Berlin Brandenburg

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  • 1
    Language: English
    In: Journal of Molecular Biology, 09 October 2013, Vol.425(19), pp.3678-3697
    Description: The RNA chaperone protein Hfq is required for the function of all small RNAs (sRNAs) that regulate mRNA stability or translation by limited base pairing in . While there have been numerous studies to characterize Hfq activity and the importance of specific residues, there has been only limited characterization of Hfq mutants . Here, we use a set of reporters as well as co-immunoprecipitation to examine 14 Hfq mutants expressed from the chromosome. The majority of the proximal face residues, as expected, were important for the function of sRNAs. The failure of sRNAs to regulate target mRNAs in these mutants can be explained by reduced sRNA accumulation. Two of the proximal mutants, D9A and F39A, acted differently from the others in that they had mixed effects on different sRNA/mRNA pairs and, in the case of F39A, showed differential sRNA accumulation. Mutations of charged residues at the rim of Hfq interfered with positive regulation and gave mixed effects for negative regulation. Some, but not all, sRNAs accumulated to lower levels in rim mutants, suggesting qualitative differences in how individual sRNAs are affected by Hfq. The distal face mutants were expected to disrupt binding of ARN motifs found in mRNAs. They were more defective for positive regulation than negative regulation at low mRNA expression, but the defects could be suppressed by higher levels of mRNA expression. We discuss the implications of these observations for Hfq binding to RNA and mechanisms of action. ► analysis of 14 chromosomally expressed mutants reveals differential consequences of specific amino acid substitutions. ► Phenotypes confirm a critical role for the proximal face of Hfq in sRNA binding. ► The rim of the Hfq hexamer is important for positive regulation by sRNAs. ► Individual sRNA:mRNA pairs show different sensitivities to mutants. ► The results suggest unexpected complexity in how Hfq promotes sRNA-based regulation.
    Keywords: Dsra ; Arcz ; Mcas ; Ryhb ; Chix ; Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 2
    Language: English
    In: Journal of Molecular Biology, 06 November 2015, Vol.427(22), pp.3491-3500
    Description: Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN) motif in , both and . These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by . Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs.
    Keywords: Hfq ; Small Non-Coding RNA ; RNA Chaperone ; RNA–Protein Interactions ; Molecular Beacon ; Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 3
    Language: English
    In: Journal of Molecular Biology, 1981, Vol.153(1), pp.67-78
    Description: The integration of bacteriophage lambda into the Escherichia coli chromosome depends on the phage-encoded Int protein; prophage excision depends on Int and a second phage function, Xis. Limited excisive recombination has been observed in vivo with certain xis mutants, suggesting that Int may be able to carry out excision without Xis. The authors report here that purified Int protein carries out lambda site-specific excisive recombination in vitro in the absence of Xis. This reaction requires host factors derived from a non-lysogenic E. coli strain and is influenced strongly by ionic strength. Excision in the absence of Xis occurs slowly at low salt concentrations (40 mm-NaCl) and very little excision occurs at high salt concentrations (100 mm-NaCl). In the presence of Xis, excisive recombination proceeds rapidly at both low and high ionic strengths. These observations are consistent with previous experiments that suggested the partial dispensability of Xis for excision.
    Keywords: Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 4
    Language: English
    In: Journal of Molecular Biology, 1980, Vol.138(3), pp.503-512
    Description: We have examined the requirements for integrative and excisive recombination of bacteriophage γ, by providing large quantities of Int and varying amounts of Xis in A and A mutant cells. The wild type A gene product is not essential for excisive recombination in the presence of large amounts of Xis. When Xis is limiting or missing, however, the A gene product plays a major role in excision. Similarly, integrative recombination is normally quite dependent on A product. Limited integrative recombination can occur in a A mutant, but only in the presence of large quantities of Xis. We propose that alternative pathways exist for these two reactions; one pathway (int, Him A) is favored for integration but can be used for excision, while the other (Int, Xis) is highly efficient for excisive recombination but can also be used for integrative recombination.
    Keywords: Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 5
    Language: English
    In: Journal of Molecular Biology, 1993, Vol.234(4), pp.1021-1037
    Description: Mutations in a number of loci, including the lon gene , dramatically increase the production of colanic acid capsular polysaccharide and render Escherichia coli K-12 mucoid. The lon gene, which encodes an ATP-dependenty protease, is localized at ten minutes on the E. coli map and is very closely linked to the hupB gene coding for one of the two subunits of the histone-like protein HU. Surprisingly the introduction of a multi-copy plasmid carrying either the hupB or hupA gene into a wild-type E. coli strain, results in the overproduction of one of the HU subunits and repression of the synthesis of the other without changing the overall concentration of HU, also renders the cells mucoid. As in a lon strain, the transcription of the cps genes, the structural genes for the synthesis of colanic acid, is induced dramatically. Protease Lon negatively regulates cps genes by destabilizing RcsA, a positive regulator of capsule synthesis. Regulation of HU synthesis does not affect the steady state level of Lon, as judged by Western blotting. The UV sensitivity of the hup transformed lon + bacteria is identical to the lon + parental strain, suggesting that Lon activity for the degradation of SulA in these cells is normal. Using lac operon fusions to cps gene promoters and to the rcsA promoter we show that the deregulation of HU synthesis does not by pass the positive regulatory action of RcsA and RcsB for the expression of cps genes but functions by stimulating RcsA synthesis.
    Keywords: Histone-Like ; Capsular Polysaccharide ; Lon Protease ; SOS Response ; Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 6
    Language: English
    In: Journal of Molecular Biology, 1983, Vol.171(3), pp.337-343
    Description: The Escherichia coli sulA gene product is a highly unstable protein, whose synthesis in response to DNA damage is associated with an inhibition of septation. Genetic evidence as well as sequence information suggests that the sulA gene is part of the E. coli SOS system and is induced after DNA damage. We have constructed a plasmid carrying only the sulA gene; this plasmid is stable only when it contains an amber mutation in the sulA structural gene. Using fragments of this plasmid, we have carried out in vitro transcription experiments and demonstrated one major start site for RNA transcription. We have mapped this initiation point to an adenylate residue 30 nucleotides before the protein start. Purified LexA protein completely abolishes this transcription, in agreement with the prediction made from the genetic and sequence information previously available.
    Keywords: Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 7
    Language: English
    In: Journal of Molecular Biology, 1982, Vol.158(3), pp.327-346
    Description: The int gene of bacteriophage lambda can be transcribed from p sub(I) in the presence of cII product or from p sub(L) in the presence of N product. On infection Int is expressed only from p sub(I), while in an induced prophage Int can be expressed from p sub(L). The critical difference is the b super(+) region, which is adjacent to int) in an infecting phage, but is separate in a prophage. When a prophage is constructed in which b super(+) is adjacent to int), the synthesis of Int under p sub(L) control is strongly inhibited. The authors have constructed lacZ-p sub(I) and lacZ-p sub(L) fusions, where the fusion is 252 base-pairs to the left of the attP site, within the b region. The expression of lacZ from p sub(L) takes place in the b53 deletion (--252 base-pairs to --160 base-pairs) but not in wild-type phage. The b53 deletion thus removes the t sub(I) terminator. It also removes the region containing the sib mutations; as expected, it permits the expression of int from p sub(L) during infection. The t sub(I) terminator is suppressed by N-function acting on transcription from p sub(L), and is active in a rho15 mutant. The cII-independent p sub(I) mutation p sub(int)c sub(226) acts as a weak promoter which is not further stimulated by cII. Since int super(c)226 is a G multiplied by C to A multiplied by T change at --11, this suggests that the interaction between p sub(I) and cII product may involve sequences in the Pribnow box region of this promoter.
    Keywords: Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 8
    Language: English
    In: Journal of Molecular Biology, 1975, Vol.91(4), pp.489-499
    Description: A sensitive assay for bacteriophage lambda site-specific recombination has been developed using the λ att 2 phage described by Shulman & Gottesman (1971). This large EDTA-sensitive transducing phage, which carries both the left and right prophage attachment sites, undergoes an Int and Xis-dependent excision reaction to yield a smaller EDTA-resistant non-transducing phage bearing a phage attachment site. We have used this system to study the characteristics of site-specific excision in vivo . We find that: (1) the kinetics of excision are rapid; (2) inhibition of macromolecular processes such as transcription, translation, and DNA replication does not affect the extent of the excisional recombination.
    Keywords: Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 9
    Language: English
    In: Journal of Molecular Biology, 1979, Vol.131(3), pp.637-649
    Description: We have studied the excision reaction of bacteriophage lambda, both in vivo and in vitro , using as a substrate a λ att 2 (L × R) phage carrying both the right and left-hand prophage attachment sites. Int and Xis are provided by induction of the heat-inducible defective prophage, λc1857 ΔH1. After a brief induction (5 min) of these cells, excisive recombination is blocked in the presence of the DNA gyrase inhibitor, coumermycin. However, after a longer induction (greater than 30 min) excisive recombination occurs efficiently under conditions where λ integrative recombination is inhibited by coumermycin. In such extensively induced coumermycin-treated cells, infecting λ att 2 (L × R) DNA is not supercoiled, and recombinants are found among the relaxed covalently closed circular DNA. In vitro , starting with a hydrogen-bonded λ att 2 DNA substrate, excision is insensitive to high concentrations of coumermycin and novobiocin. To study the DNA substrate requirements for excisive recombination in more detail, we have developed a restriction fragment assay for excisive recombination. With this assay, we demonstrate that supercoiled, hydrogen-bonded, and linear λ att 2 DNA molecules are all efficient substrates in the in vitro excision reaction. Spermidine is required but ATP and Mg 2+ are not. We conclude that supercoiling is not an absolute requirement for site-specific recombination of λ.
    Keywords: Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 10
    Language: English
    In: Journal of Molecular Biology, 1969, Vol.44(1), pp.117-127
    Description: An F- thr-ara ‡ ‡ Abbreviations used: lac , ara , gal , utilization of lactose, arabinose, galactose; his , pyr , pur , arg , thr , leu , pro , trp requirement for histidine, pyrimidine, purine, arginine, threonine, leucine, proline, tryptophan; T1 r , T1,5 r , resistance to phage T1, phages T1 and T5; att 80, chromosome attachment site for φ80; Sm s , Sm r , sensitive and resistant to streptomycin; φ80d ara , φ80d lac , defective φ80 transducing phage carrying the ara region, the lac region; F TS , F factor with mutation rendering its replication temperature sensitive; φ80 h , φ80 host range; φ80 v , φ80 virulent; λ hφ80, λ—φ80 hybrid having λ immunity and φ80 host range and attachment specificity. episome has been inserted into the Escherichia coli chromosome at the T1 locus near the attachment site for bacteriophage φ80. From a strain carrying such an insertion, a φ80d ara transducing phage was isolated. The techniques used should be applicable for the isolation of specialized transducing phages for many E. coli genes.
    Keywords: Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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