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  • 1
    Electronic Resource
    Electronic Resource
    Palo Alto, Calif. : Annual Reviews
    Annual Review of Biochemistry 62 (1993), S. 749-797 
    ISSN: 0066-4154
    Source: Annual Reviews Electronic Back Volume Collection 1932-2001ff
    Topics: Chemistry and Pharmacology , Biology
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 4 (1990), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: From the effects of 13 deletions and three linker-scanner mutations at the Escherichia coli nirB promoter we have located sequences necessary for FNR-dependent induction of activity by anaerobiosis and further nitrite-dependent stimulation of expression. We describe a nirB promoter derivative that allows the cloning of ‘cassettes’ carrying different FNR-binding sequences and experiments in which a number of point mutations were introduced into these sequences. FNR-dependent stimulation of expression from the nirB promoter is critically dependent on the location of the FNR-binding site, and deletion or insertion of one base pair is sufficient to disrupt promoter function. We have transferred a number of cassette FNR-binding sequences from the nirB promoter to the unrelated melR promoter. The insertion of FNR-binding sequences at the melR promoter is sufficient to confer fnr-dependency on expression. However expression from these hybrid promoters is not as efficiently repressed during aerobic growth, suggesting that the function of bound FNR is dependent on the sequence context of the FNR-binding sequence.
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Expression from both the Escherichia coli nir and nrf promoters is dependent on anaerobic induction by FNR but is further regulated by NarL and NarP in response to the presence of nitrite and nitrate in the growth medium. The nir promoter is activated by NarL in response to nitrate and nitrite and activated by NarP in response to nitrate but not nitrite. The effects of point mutations suggest that NarL and NarP both bind to the same target, which is a pair of heptamer sequences organized as an inverted repeat, centred 691/2 bp upstream of the transcript startpoint. The nrf promoter can be activated by either NarP or NarL in response to nitrite but is repressed by NarL in response to nitrate. Mutational analysis of the nrf promoter has been exploited to corroborate the location of the -10 hexamer and the FNR-binding site, and to find the sites essential for nitrite-dependent activation and nitrate-dependent repression. Optimal activation by NarP or NarL in response to nitrite requires an inverted pair of heptamer sequences, similar to that found at the nir promoter, but centred 741/2 bp upstream from the transcript start. NarL-dependent repression by nitrate is due to two heptamer sequences that flank the FNR-binding sequence. We conclude that NarL and NarP bind to the same heptamer sequences, but that the affinities for the two factors vary from site to site.
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  • 4
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In Escherichia coli, FNR and CRP are homologous transcriptional regulators which recognize similar nucleotide sequences via DNA-binding domains containing analogous helix-turn-helix motifs. The molecular basis for recognition and discrimination of their target sites has been investigated by directed amino acid substitutions in the corresponding DNA-recognition helices. In FNR, Glu-209 and Ser-212 are essential residues for the recognition of FNR sites. A V208R substitution confers CRP-site specificity without loss of FNR specificity, but this has adverse effects on anaerobic growth. In contrast, changes at two (V206R and E209D) or three (V208R, S212G and G216K) positions in FNR endow a single CRP-site binding specificity. In reciprocal experiments, two substitutions (R180V and G184S) were required to convert the binding specificity of CRP to that of FNR. Altering Asp-199 in FNR failed to produce a positive control phenotype, unlike substitutions at the comparable site in CRP. Implications for the mechanism of sequence discrimination by FNR and CRP are discussed.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Transcription initiation at the Escherichia coli nirB promoter is induced by anaerobic growth and further increased by the presence of nitrite or nitrate in the growth medium. Expression from this promoter is totally dependent on the transcription factor, FNR, which binds between positions −52 and −30 upstream of the transcription startsite. The 20 base pairs from position −79 to −60 contain an inverted repeat of two 10-base sequence elements that are related to sequences at the NarL-binding site at the E. coli narG promoter. Comparison of these, and sequence elements at other promoters regulated by NarL, suggests a consensus NarL-binding sequence. Mutations in the putative NarL-binding site at the nirB promoter decrease FNR-dependent anaerobic induction, suggesting that NarL acts as a helper to FNR during transcription activation. These mutations also suppress induction by nitrite: single mutations at symmetry-related positions have similar effects, whilst double mutations have more severe effects, probably because two NarL subunits bind to the inverted repeat. Disruption of narL decreases nitrite induction of the nirB promoter whilst not suppressing induction by nitrate, suggesting that there may be a second nitrate-responsive factor. Nitrate induction was, however, suppressed by double mutations at symmetry-related positions in the NarL-binding site, suggesting that this putative second factor may bind to sequences similar to those recognized by NarL.
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  • 6
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Using recombinant DNA techniques, nested deletions have been made upstream of the Escherichia coli nirB transcription start site and their effects on the regulation of nirB promoter activity have been measured. Nucleotide sequences downstream of −73 are sufficient for FNR-dependent induction of activity by anaerobic growth conditions. However, nucleotide sequences between −87 and −149 are essential for further induction by nitrite in the growth medium. The nucleotide sequence at the galP1 CRP binding site located from −31 to −52 displays some similarities with the same region at the nirB promoter. When the galP1 sequence from −30 to −54 was replaced by the corresponding nirB sequence, expression from galP1 became inducible by FNR under anaerobic growth conditions.
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 2 (1988), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Starting with a DNA fragment containing the galactose operon P 2 promoter, we made a series of deletions that progressively replaced DNA sequences upstream of the transcription startpoint and determined their effects on P 2 activity. The results show that specific sequences upstream of −32 are not important. Removal of the sequence 5′-CACA-3′ from −32 to −28 reduces P 2 activity by 50%: longer deletions to −16 further reduce activity but do not remove the information specifying the transcription startpoint. DNA sequences between −32 and −16 at galP 2 assist the isomerization of RNA polymerase from closed to open complexes rather than contributing to the initial binding of RNA polymerase. The activity of gal P 2 in the absence of −35 region sequences is dependent on the sequence TG just upstream of the − 10 hexamer, TATACT: a mutation at −14 changing the TG sequence to TT totally inactivates P 2. However, P 2 activity can be restored if the consensus −35 region sequence TTGACA is cloned 17 bp upstream of the −10 hexamer. Thus, for transcription initiation, the −10 hexamer, TATACT, must ‘cooperate’ with upstream sequences that may be located either around −35 or −14.
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  • 8
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Using gene-manipulation techniques, we made a set of short insertions and deletions in the Escherichia coli galactose operon between the transcription start site and the Shine-Dalgarno sequence of the first gene of the operon, galE. Translation initiation is severely reduced when the distance between the 5′ end of the message and the Shine-Daigarno sequence drops below 15 bases.Transcription of the gal operon can start at two distinct sites, S1 and S2, separated by 5 bp, situated 16 and 21 bp upstream of the galE Shine-Dalgarno sequence, respectively. When transcription starts at S2, gal operon expression is discoordinate as the galE gene is better translated than promoter-distal genes. Here we report that gal operon expression is discoordinate even when message starting at S2 is shortened. This shortened. that the better translation of galE from transcripts starting at S2 is not simply due to the fact that they are longer than transcripts starting at S1.
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  • 9
    ISSN: 0378-1119
    Keywords: Plasmid expression vector ; gal regulatory region ; lac gene ; promoter consensus sequences ; twist in spacer region
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Gene 59 (1987), S. 253-263 
    ISSN: 0378-1119
    Keywords: Recombinant DNA ; deletion analysis ; divergent promoter ; melibiose-dependent transcription ; nucleotide sequence analysis ; positive control of transcription ; transcript mapping
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
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