The C-Terminal 12 Amino Acids of ςN Are Required for Structure and Function

Studholme, David J.; Finn, Robert D.; Chaney, Matthew; Buck, Martin

doi:10.1006/abbi.1999.1426

Cited by 3 publications

(4 citation statements)

References 22 publications

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“…1, lanes 2 and 6). Also, whereas the K. pneumoniae N -E. coli core complex ran as a single band on the gel, the A. aeolicus N -E. coli core complex ran as at least two discrete bands, suggesting more than one conformation, a phenomenon that has previously been reported for several mutant K. pneumoniae N -E. coli core complexes (45,54). Sequence-specific DNA-binding activity of A. aeolicus N .…”

Section: Resultssupporting

confidence: 59%

“…The special form of bacterial RNA polymerase (RNAP) containing alternative sigma factor N or 54 ( N -RNAP), initiates transcription by a mechanism quite distinct from RNAP containing the major 70 sigma factor (35,39,48). Transcription initiation by N -RNAP requires the hydrolysis of nucleoside triphosphate, catalyzed by activator proteins bound to upstream activator sequences (enhancer elements).…”

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Functionality of Purified ς ^N (ς ⁵⁴ ) and a NifA-Like Protein from the Hyperthermophile Aquifex aeolicus

et al. 2000

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The genome sequence of the extremely thermophilic bacterium Aquifex aeolicus encodes alternative sigma factor N ( 54 , RpoN) and five potential N -dependent transcriptional activators. Although A. aeolicus possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria. We identified five putative N -dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity. We cloned, overexpressed (in Escherichia coli), and purified A. aeolicus N and the NifA homologue, AQ_218. Purified A. aeolicus N bound to E. coli core RNA polymerase and bound specifically to a DNA fragment containing E. coli promoter glnHp2 and to several A. aeolicus DNA fragments containing putative N -dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicus N supported A. aeolicus NifA-dependent transcription from the glnHp2 promoter. The E. coli activator PspF⌬HTH did not stimulate transcription. The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the A. aeolicus glnBA operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism. These results argue that the N enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and N have coevolved.The special form of bacterial RNA polymerase (RNAP) containing alternative sigma factor N or 54 ( N -RNAP), initiates transcription by a mechanism quite distinct from RNAP containing the major 70 sigma factor (35,39,48). Transcription initiation by N -RNAP requires the hydrolysis of nucleoside triphosphate, catalyzed by activator proteins bound to upstream activator sequences (enhancer elements). The mechanisms of regulation involving this form of RNAP are among the most sophisticated in bacteria (52).Since this novel form of RNAP was first recognized in enteric bacteria (25,29,47), N has been discovered in many other bacteria, including several proteobacteria, as well as in the gram-positive Bacillus subtilis (14) and in Planctomyces limnophila (36). Furthermore, N appears to be encoded by the genomes of the hyperthermophile Aquifex aeolicus (15), the spirochete Borrelia burgdorferi (18), and the obligate intracellular pathogens Chlamydia trachomatis (51) and Chlamydia pneumoniae (31).The N proteins from Klebsiella pneumoniae and Escherichia coli have been the subject of much genetic and biochemical analysis (e.g., see references 8, 20, and 23). Although considerable progress has been made towards understanding the mechanisms by which N performs its function, to date no high-resolution structural data are available for this system. Thermophilic homologues of mesophilic proteins have often been shown to be tractable for structural studies. For example, the crystal structures of the histidine kinase domain of CheA from Thermotoga maritima and the core RNAP from...

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Section: Resultssupporting

confidence: 59%

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confidence: 99%

Functionality of Purified ς ^N (ς ⁵⁴ ) and a NifA-Like Protein from the Hyperthermophile Aquifex aeolicus

et al. 2000

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“…This indicates that ‘leaky’ expression of fliA was sufficient to restore fliA ‐dependent motility. We have previously observed ‘leaky’ expression of genes encoding sigma factors from pET‐derived plasmids in non‐DE3 strains [15,16]. The reason for the difference in complementation between the two plasmid vectors is difficult to pinpoint but may be related to copy number and/or levels of expression of the protein.…”

Section: Resultsmentioning

confidence: 99%

The alternative sigma factor Ï²⁸of the extreme thermophileAquifex aeolicusrestores motility to anEscherichia coli fliAmutant

Studholme

Buck

2000

FEMS Microbiology Letters

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Sigma factor sigma(28) (sigma(F), FliA, SigD) directs RNA polymerase to transcribe the genes required for flagellar biosynthesis and chemotaxis in many bacteria, including Bacillus subtilis, Legionella pneumophila, Salmonella typhimurium, Escherichia coli, Yersinia enterolytica, Treponema maltophilum and Pseudomonas aeruginosa. Remarkably the fliA gene from the extreme thermophile Aquifex aeolicus restored motility to the E. coli mutant at relatively low temperature, albeit partially. This clearly demonstrates that A. aeolicus sigma(28) is able to direct RNA polymerase to E. coli sigma(28)-dependent promoters and take part in the complex interactions required to support transcription of the flagellar apparatus in vivo. The ability of A. aeolicus sigma(28) to function with mesophilic components shows that critical functional interactions made by these sigma factors are well conserved, and are not dependent upon high temperature. We over-produced and purified the sigma(28) protein and demonstrated binding to E. coli core RNA polymerase in vitro. In common with SigD from B. subtilis, but unlike most sigma factors, A. aeolicus sigma(28) showed DNA binding activity in vitro but there was no evidence of sequence specificity. We note that A. aeolicus sigma(28) is a good candidate for structural studies.

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“…This indicates that leaky' expression of £iA was su¤cient to restore £iA-dependent motility. We have previously observed`leaky' expression of genes encoding sigma factors from pET-derived plasmids in non-DE3 strains [15,16]. The reason for the di¡erence in complementation between the two plasmid vectors is di¤cult to pinpoint but may be related to copy number and/or levels of expression of the protein.…”

Section: A Aeolicus £Ia Complements E Coli £Ia Mutant In Vivomentioning

confidence: 99%

The alternative sigma factor σ28 of the extreme thermophile Aquifex aeolicus restores motility to an Escherichia coli fliA mutant

Studholme¹

2000

FEMS Microbiology Letters

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Sigma factor c 28 (c F , FliA, SigD) directs RNA polymerase to transcribe the genes required for flagellar biosynthesis and chemotaxis in many bacteria, including Bacillus subtilis, Legionella pneumophila, Salmonella typhimurium, Escherichia coli, Yersinia enterolytica, Treponema maltophilum and Pseudomonas aeruginosa. Remarkably the fliA gene from the extreme thermophile Aquifex aeolicus restored motility to the E. coli mutant at relatively low temperature, albeit partially. This clearly demonstrates that A. aeolicus c 28 is able to direct RNA polymerase to E. coli c 28 -dependent promoters and take part in the complex interactions required to support transcription of the flagellar apparatus in vivo. The ability of A. aeolicus c 28 to function with mesophilic components shows that critical functional interactions made by these sigma factors are well conserved, and are not dependent upon high temperature. We over-produced and purified the c 28 protein and demonstrated binding to E. coli core RNA polymerase in vitro. In common with SigD from B. subtilis, but unlike most sigma factors, A. aeolicus c 28 showed DNA binding activity in vitro but there was no evidence of sequence specificity. We note that A. aeolicus c 28 is a good candidate for structural studies. ß

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The C-Terminal 12 Amino Acids of ςN Are Required for Structure and Function

Cited by 3 publications

References 22 publications

Functionality of Purified ς ^N (ς ⁵⁴ ) and a NifA-Like Protein from the Hyperthermophile Aquifex aeolicus

Functionality of Purified ς ^N (ς ⁵⁴ ) and a NifA-Like Protein from the Hyperthermophile Aquifex aeolicus

The alternative sigma factor Ï²⁸of the extreme thermophileAquifex aeolicusrestores motility to anEscherichia coli fliAmutant

The alternative sigma factor σ28 of the extreme thermophile Aquifex aeolicus restores motility to an Escherichia coli fliA mutant

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The C-Terminal 12 Amino Acids of ςN Are Required for Structure and Function

Cited by 3 publications

References 22 publications

Functionality of Purified ς N (ς 54 ) and a NifA-Like Protein from the Hyperthermophile Aquifex aeolicus

Functionality of Purified ς N (ς 54 ) and a NifA-Like Protein from the Hyperthermophile Aquifex aeolicus

The alternative sigma factor Ï28of the extreme thermophileAquifex aeolicusrestores motility to anEscherichia coli fliAmutant

The alternative sigma factor σ28 of the extreme thermophile Aquifex aeolicus restores motility to an Escherichia coli fliA mutant

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Functionality of Purified ς ^N (ς ⁵⁴ ) and a NifA-Like Protein from the Hyperthermophile Aquifex aeolicus

Functionality of Purified ς ^N (ς ⁵⁴ ) and a NifA-Like Protein from the Hyperthermophile Aquifex aeolicus

The alternative sigma factor Ï²⁸of the extreme thermophileAquifex aeolicusrestores motility to anEscherichia coli fliAmutant