In vivo function of Escherichia coli pyruvate oxidase specifically requires a functional lipid binding site

ABSTRACT FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position −41.5 relative to the transcription start site. A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo. FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions. However, sharp repression is observed when the upstream-bound FNR is located near positions −85 or −95. This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the yfiD promoter. A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position −61.5 and a second upstream DNA site for FNR, was also constructed. Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions.

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Studies of the thiamin pyrophosphate binding site of Escherichia coli pyruvate oxidase. Evidence for an essential tryptophan residue.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)85699-5 ◽

1980 ◽

Vol 255 (8) ◽

pp. 3302-3307

Author(s):

T.A. O'Brien ◽

R.B. Gennis

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Tryptophan Residue ◽

Pyruvate Oxidase ◽

Thiamin Pyrophosphate

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Identification of the Lipid-Binding Site of Phosphatidylcholine-Transfer Protein with Phosphatidylcholine Analogs Containing Photactivable Carbene Precursors

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1983.tb07382.x ◽

1983 ◽

Vol 132 (2) ◽

pp. 441-449 ◽

Cited By ~ 38

Author(s):

Jan WESTERMAN ◽

Karel W. A. WIRTZ ◽

Theo BERKHOUT ◽

Laurens L. M. DEENEN ◽

Ramachandran RADHAKRISHNAN ◽

...

Keyword(s):

Binding Site ◽

Lipid Binding ◽

Transfer Protein ◽

Phosphatidylcholine Transfer Protein ◽

Lipid Binding Site

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Phospholipid-induced structural changes to an erythroid β spectrin ankyrin-dependent lipid-binding site

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2008.07.020 ◽

2008 ◽

Vol 1778 (11) ◽

pp. 2612-2620 ◽

Cited By ~ 13

Author(s):

Aleksander Czogalla ◽

Krzysztof Grzymajło ◽

Adam Jezierski ◽

Aleksander F. Sikorski

Keyword(s):

Binding Site ◽

Structural Changes ◽

Lipid Binding ◽

Lipid Binding Site

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Alterations in the binding site of the cyclic AMP receptor protein at the Escherichia coli galactose operon regulatory region

Biochemical Journal ◽

10.1042/bj2530809 ◽

1988 ◽

Vol 253 (3) ◽

pp. 809-818 ◽

Cited By ~ 10

Author(s):

K Gaston ◽

B Chan ◽

A Kolb ◽

J Fox ◽

S Busby

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Binding Site ◽

Consensus Sequence ◽

Regulatory Region ◽

Receptor Protein ◽

Binding Assays ◽

Cyclic Amp Receptor Protein ◽

Stimulation Of

Gene manipulation techniques have been used to alter the binding site for the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP) at the regulatory region of the Escherichia coli galactose (gal) operon. The effects of these changes on CRP-dependent stimulation of expression from the galP1 promoter in vivo have been measured, and gel binding assays have been used to measure the affinity of cAMP-CRP for the modified sites. Firstly we have deleted progressively longer sequences from upstream of the gal CRP site in order to locate the functional limit of the site. A deletion to -49, removing the first base that corresponds to the consensus sequence for a CRP binding site, is sufficient to reduce CRP binding and block CRP-dependent stimulation of P1. Secondly, we used synthetic oligonucleotides to invert the asymmetric nucleotide sequence at the gal CRP binding site or to make the sequence symmetric. Inversion of the site has little effect on CRP binding, the architecture of open complexes at P1 revealed by DNAase I footprinting, or the stimulation of transcription from P1. Making the site symmetric increases the affinity for CRP by over 50-fold and leads to increased transcription from P1, whilst hardly altering the DNAase I footprint of open complexes. Our results confirm that the strength of binding of CRP depends on the nature of the site and show that it is this that principally accounts for differences in CRP-dependent stimulation of transcription.

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