Characterization of the Escherichia coli araFGH and araJ promoters

Hendrickson, William; Stoner, Carol M.; Schleif, Robert

doi:10.1016/s0022-2836(05)80163-9

Cited by 32 publications

(41 citation statements)

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“…2 and 3). Searches of the protein data bases with the deduced protein sequence of this downstream ORF revealed significant homology with only one protein, namely, the XylR protein of Bacillus subtilis, which is involved in regulation of the xylose operon (19 (11) and that this transcription is regulated by CAP and AraC (11,18) are, however, consistent with araJ actually being translated and serving some purpose in the arabinose metabolism of E. coli. Furthermore, if araJ had been without physiological value for an extended period of evolutionary time, we would expect it to have acquired nonsense mutations, terminating translation well before the REPs.…”

Section: Resultsmentioning

confidence: 94%

“…3. Summary of sequence and footprinting (11) data. The DNA sequence is numbered relative to the araJ transcriptional start site.…”

Section: Resultsmentioning

confidence: 99%

“…Recent work (11,18) has shown that arabinose induces substantial transcription from pJ in vivo and in vitro. In vivo, as well as in vitro (11,18), transcription from pJ is dependent upon the arabinosespecific transcriptional regulator protein AraC and the global catabolite gene activator protein CAP, as is transcription from the araBAD, araE, and araFGH operons (7,8,32).Since all known ara mutations are confined to the araBAD, araC, araE, and araFGH operons, we were interested to learn whether pJ drives an intact gene and whether its product plays any detectable role in vivo. In particular, we wished to determine whether the araJ product was a fourth component in the high-affinity arabinose transport system, since most analogous systems contain four protein components (1, 34).…”

mentioning

confidence: 99%

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confidence: 99%

“…This promoter was originally cloned by Kosiba and Schleif (18) and at that time was misidentified as the araFGH promoter. Subsequent work (11,13) has identified the actual araFGH promoter, and the arabinose-inducible promoter cloned by Kosiba and Schleif (18) is now known as pJ. Recent work (11,18) has shown that arabinose induces substantial transcription from pJ in vivo and in vitro.…”

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confidence: 99%

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Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon

Reeder

Schleif

1991

J Bacteriol

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We report the mapping, sequencing, and study of the physiological role of the fourth arabinose-inducible operon from Escherichia coli, araJ. It is located at 9 min on the chromosome and codes for a single 42-kDa protein that shows no significant homology to other known proteins. Destruction of the chromosomal araJ gene does not detectably affect either of the two arabinose transport systems, the ability of cells to grow The enteric bacterium Escherichia coli can utilize L-arabinose as a sole carbon source. In the presence of arabinose, expression from the three known ara operons is induced up to 300-fold. These are the araBAD operon (5, 6), which codes for proteins involved in the catabolism of arabinose, and the araE (27) and araFGH (2, 17, 31) operons, which code for the low-and high-affinity arabinose transport systems, respectively: In addition to the promoters of these three well-characterized operons, there exists a fourth promoter in E. coli that is induced by arabinose. This promoter was originally cloned by Kosiba and Schleif (18) and at that time was misidentified as the araFGH promoter. Subsequent work (11, 13) has identified the actual araFGH promoter, and the arabinose-inducible promoter cloned by Kosiba and Schleif (18) is now known as pJ. Recent work (11,18) has shown that arabinose induces substantial transcription from pJ in vivo and in vitro. In vivo, as well as in vitro (11,18), transcription from pJ is dependent upon the arabinosespecific transcriptional regulator protein AraC and the global catabolite gene activator protein CAP, as is transcription from the araBAD, araE, and araFGH operons (7,8,32).Since all known ara mutations are confined to the araBAD, araC, araE, and araFGH operons, we were interested to learn whether pJ drives an intact gene and whether its product plays any detectable role in vivo. In particular, we wished to determine whether the araJ product was a fourth component in the high-affinity arabinose transport system, since most analogous systems contain four protein components (1, 34).Here we report the mapping of the araJ gene to 9 min on the E. coli chromosome and the cloning and sequencing of the araJ gene and surrounding regions of the chromosome. We also show that insertion/deletion mutations in araJ have no detectable effect on the ability of the bacteria to grow on arabinose and to induce the araBAD operon. MATERIALS AND METHODSMedia, strains, and general methods. Media and general methods have been described (21,33 Kosiba and Schleif (18) containing the araJ promoter. Three positive clones were selected and shown by restriction mapping to contain overlapping copies of the same piece of genomic DNA; these three clones were sent to us for further analysis.One of the lambda clones was digested with PvuI, and the 1.9-and 2.5-kb fragments that contained pJ and flanking sequences were isolated. The fragments were filled out with DNA polymerase (Klenow fragment) and then ligated into 7765 JOURNAL OF BACTERIOLOGY, Dec. 1991, p. 7765-7771

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Section: Resultsmentioning

confidence: 94%

“…3. Summary of sequence and footprinting (11) data. The DNA sequence is numbered relative to the araJ transcriptional start site.…”

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon

Reeder

Schleif

1991

J Bacteriol

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Elucidating residue roles in engineered variants of AraC regulatory protein

Tang

Cirino

2010

Protein Science

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The AraC regulatory protein was previously engineered to control gene expression specifically in response to D-arabinose and not the native effector L-arabinose (Tang et al., J Am Chem Soc 2008;130:5267-5271). Mutations were targeted in the ligand-binding pocket and on the AraC N-terminal arm, which plays an important role in maintaining repressing or activating conformations in the absence or presence of effector, respectively. In this study, we analyze the contributions of individual mutations toward the overall mutant functions in an attempt to streamline future AraC design efforts. For a variety of point mutants, we quantify the induced expression response to D-arabinose (level of leaky expression, induction fold, half-maximal dose response, and effector specificity) and the binding affinity of the purified ligand-binding domain for D-arabinose. We find that mutations introduced in the N-terminal arm (design Position 8) strengthen the induction response, most likely by weakening interactions with the DNA-binding domain, but are not involved in ligand binding. Meanwhile, binding pocket mutations occurring further away from the arm (Positions 80 and 82) primarily contribute to maintaining repression in the absence of effector and do not show response to D-arabinose without the accompanying mutations. Combinations of mutations cooperatively couple molecular recognition to transcriptional activation, demonstrating the complexity of the AraC regulatory switch and the power of combinatorial protein design to alter effector specificity while maintaining regulatory function.

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The CAP Modulon

Busby¹,

Kolb²

1996

Regulation of Gene Expression in Escherichia Coli

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Characterization of the Escherichia coli araFGH and araJ promoters

Cited by 32 publications

References 35 publications

Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon

Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon

Elucidating residue roles in engineered variants of AraC regulatory protein

The CAP Modulon

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