Three-dimensional crystal structures of Escherichia coli met repressor with and without corepressor

Rafferty, John B.; Somers, W.S.; Saint‐Girons, Isabelle; Phillips, Simon E. V.

doi:10.1038/341705a0

Cited by 181 publications

(132 citation statements)

References 27 publications

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“…These results suggested that a complex of MetJ and SAM binds a common nucleotide sequence in the promoters of target genes to prevent their transcription (Greene et al, , 1973Hobson & Smith, 1973). Identification of a consensus repressor binding site ('met box'; Belfaiza et al, 1986) and crystallographic studies (Rafferty et al, 1989) all support this model. Genes most induced by defects in either metJ or metK include metA, B, C, E, F, K, L (as the downstream gene in the metBL operon) and metR (a regulatory protein that, by itself or when bound with homocysteine, regulates expression of a variety of genes in the met regulon to coordinate the activities of the two branches of the methionine biosynthetic pathway; again, see Greene, 1996).…”

Section: Introductionsupporting

confidence: 48%

In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli

LaMonte

Hughes

2006

Microbiology

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Regulation of methionine biosynthesis in Escherichia coli involves a complex of the MetJ aporepressor protein and S-adenosylmethionine (SAM) repressing expression of most genes in the met regulon. To test the role of SAM in the regulation of met genes directly, SAM pools were depleted by the in vivo expression of the cloned plasmid vector-based coliphage T3 SAM hydrolase (SAMase) gene. Cultures with in vivo SAMase activity were assayed for expression of the metA, B, C, E, F, H, J, K and R genes in cells grown in methionine-rich complete media as well as in defined media with and without L-methionine. In vivo SAMase activity dramatically induced expression between 11-and nearly 1000-fold depending on the gene assayed for all but metJ and metH, and these genes were induced over twofold. metJ : : Tn5 (aporepressor defective) and metK : : Tn5 (SAM synthetase impaired; produces <5 % of wild-type SAM) strains containing in vivo SAMase activity produced even higher met gene activity than that seen in comparably prepared cells with wild-type genes for all but metJ in a MetJ-deficient background. The SAMasemediated hyperinduction of metH in wild-type cells and of the met genes assayed in metJ : : Tn5 and metK : : Tn5 cells provokes questions about how other elements such as the MetR activator protein or factors beyond the met regulon itself might be involved in the regulation of genes responsible for methionine biosynthesis.

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Section: Introductionsupporting

confidence: 48%

In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli

LaMonte

Hughes

2006

Microbiology

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“…This prediction holds for all papillomavirus E2 proteins sequenced to date. Although there is no primary sequence similarity between the 333-344 DNA contact region and the DNA contacting p-strand portions of the Met or Arc repressors (Rafferty et al 1989;Breg et al 1990), we suspect that E2 may fold similarly at its point of interaction with DNA. Although these models suggest folding as extended peptide, an a-helical conformation is also possible as proline is absent from this region.…”

Section: Discussionmentioning

confidence: 99%

Amino acids necessary for DNA contact and dimerization imply novel motifs in the papillomavirus E2 trans-activator.

et al. 1992

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The bovine papillomavirus E2 protein regulates viral transcription by binding as a dimer to the DNA sequence ACCGN4CGGT. The dimerization and DNA-binding properties are localized within its carboxy-terminal 85 amino acids (325-410). Utilizing random mutagenesis coupled with phenotypic selection in yeast, functionally important amino acids in the DNA-binding domain were identified. Four trans-activation defective point mutants within a short segment (amino acids 337-344) were DNA binding defective but dimeric. The mutation of a conserved tryptophan to serine also eliminated DNA binding, but loss of dimerization was implicated because addition of dimeric monoclonal antibody complemented this defect. A simple assay for £2 dimerization was developed using UV irradiation to produce an interchain cross-link within a dimer. No heterodimeric complexes were formed when pools of E2 of varying lengths were mixed, and only proteins with tryptophan at position 360 could be UV cross-linked. Peptide mapping of irradiated E2 protein localized the cross-link to an 18-amino-acid region bracketing this tryptophan. Substitutions for this tryptophan demonstrated the requirement for a hydrophobic residue at this position, but surprisingly, even alanine was functional. Replacement of this tryptophan with three polar amino acids or glycine eliminated DNA-binding activity, but addition of dimeric monoclonal antibody restored this function. The amino acids that were identified as being involved in DNA contact and dimerization imply that these functions are mediated by novel binding motifs.

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“…Members of the RHH family are much smaller proteins, ranging from the 45-residue CopG repressor to the 133-residue NikR from E. coli, with variations caused by additional N-or C-terminal domains (17,30). Examples of C-terminal functional regions in RHH members include protein tetramerization in the Mnt repressor, binding of the corepressor S-adenosylmethionine in MetJ, binding of the ParE toxin protein in the ParD repressor, and nickel binding by the repressor NikR (31)(32)(33)(34)(35)(36). In NikR, a C-terminal nickel-binding domain is attached to an N-terminal RHH fold (36).…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

Zhou

Kallhoff

et al. 2004

Journal of Biological Chemistry

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The PutA flavoprotein from Escherichia coli is a transcriptional repressor and a bifunctional enzyme that regulates and catalyzes proline oxidation. PutA represses transcription of genes putA and putP by binding to the control DNA region of the put regulon. The objective of this study is to define and characterize the DNA binding domain of PutA. The DNA binding activity of PutA, a 1320 amino acid polypeptide, has been localized to N-terminal residues 1-261. After exploring a potential DNA-binding region and an N-terminal deletion mutant of PutA, residues 1-90 (PutA90) were determined to contain DNA binding activity and stabilize the dimeric structure of PutA. Cell-based transcriptional assays demonstrate that PutA90 functions as a transcriptional repressor in vivo. The dissociation constant of PutA90 with the put control DNA was estimated to be 110 nM, which is slightly higher than that of the PutA-DNA complex (K d ϳ 45 nM). Primary and secondary structure analysis of PutA90 suggested the presence of a ribbonhelix-helix DNA binding motif in residues 1-47. To test this prediction, we purified and characterized PutA47. PutA47 is shown to purify as an apparent dimer, to exhibit in vivo transcriptional activity, and to bind specifically to the put control DNA. In gel-mobility shift assays, PutA47 was observed to bind cooperatively to the put control DNA with an overall dissociation constant of 15 nM for the PutA47-DNA complex. Thus, Nterminal residues 1-47 are critical for DNA-binding and the dimeric structure of PutA. These results are consistent with the ribbon-helix-helix family of transcription factors.

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Three-dimensional crystal structures of Escherichia coli met repressor with and without corepressor

Cited by 181 publications

References 27 publications

In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli

In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli

Amino acids necessary for DNA contact and dimerization imply novel motifs in the papillomavirus E2 trans-activator.

Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

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