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Article: Interaction of a putative transcriptional regulatory protein and the thermo-inducible cts-52 mutant repressor in the Bacillus subtilis phage φ105 genome

TitleInteraction of a putative transcriptional regulatory protein and the thermo-inducible cts-52 mutant repressor in the Bacillus subtilis phage φ105 genome
Authors
KeywordsBacillus subtilis
Bacteriophage φ105
cts-52 repressor
Reverse transcriptase polymerase chain reaction
Surface plasmon resonance
Issue Date2003
PublisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/jmb
Citation
Journal Of Molecular Biology, 2003, v. 333 n. 1, p. 21-31 How to Cite?
AbstractA 144 amino acid residue cts-52 mutant repressor (mtcφ105) located in the EcoRI-F immunity region (immF) of Bacillus subtilis phage φ105 is involved in the control mechanism of a thermo-inducible expression system. Adjacent to the repressor gene, an open-reading frame, designated ORF4, encodes a polypeptide of 90 amino acid residues, which shares a 37% homology with the amino acid sequence of the repressor. On the basis of the protein sequence alignment, a DNA-binding α helix-β turn-α helix (HTH) motif was identified in the N-terminal region (residues 18-37) of the repressor as well as in the polypeptide of ORF4 (residues 22-41). In vivo expression of the mutant repressor and ORF4 were confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. To study their DNA binding properties, the wild-type repressor (wtcφ105) and the mutant repressor mtcφ105, which has a Thr17 to Ile substitution, were overexpressed in Escherichia coli and purified for affinity assays. Their affinities towards six operator sites at various temperatures were elucidated by surface plasmon resonance (SPR). Our data showed that a temperature shift does not influence the wtcφ105-operators' binding affinity, while the binding of mtcφ105 to the operators was temperature sensitive. This explains how thermo-induction triggers the release of the mutant repressor and renders heterologous gene expression. Interestingly, mtcφ105 and ORF4 demonstrated a large affinity discrepancy towards individual operators at different temperatures. mRNA levels monitored by real-time RT-PCR indicated a suppression of mtcφ105 expression, but a stimulation of ORF4 transcription after thermo-induction. Our data suggested that ORF4 might be a counter protein to the phage repressor in the modulation of the two divergent-oriented promoters P M and P R within the immF region. © 2003 Elsevier Ltd. All rights reserved.
Persistent Identifierhttp://hdl.handle.net/10722/54339
ISSN
2015 Impact Factor: 4.517
2015 SCImago Journal Rankings: 3.002
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorChan, AYen_HK
dc.contributor.authorLim, BLen_HK
dc.date.accessioned2009-04-03T07:43:49Z-
dc.date.available2009-04-03T07:43:49Z-
dc.date.issued2003en_HK
dc.identifier.citationJournal Of Molecular Biology, 2003, v. 333 n. 1, p. 21-31en_HK
dc.identifier.issn0022-2836en_HK
dc.identifier.urihttp://hdl.handle.net/10722/54339-
dc.description.abstractA 144 amino acid residue cts-52 mutant repressor (mtcφ105) located in the EcoRI-F immunity region (immF) of Bacillus subtilis phage φ105 is involved in the control mechanism of a thermo-inducible expression system. Adjacent to the repressor gene, an open-reading frame, designated ORF4, encodes a polypeptide of 90 amino acid residues, which shares a 37% homology with the amino acid sequence of the repressor. On the basis of the protein sequence alignment, a DNA-binding α helix-β turn-α helix (HTH) motif was identified in the N-terminal region (residues 18-37) of the repressor as well as in the polypeptide of ORF4 (residues 22-41). In vivo expression of the mutant repressor and ORF4 were confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. To study their DNA binding properties, the wild-type repressor (wtcφ105) and the mutant repressor mtcφ105, which has a Thr17 to Ile substitution, were overexpressed in Escherichia coli and purified for affinity assays. Their affinities towards six operator sites at various temperatures were elucidated by surface plasmon resonance (SPR). Our data showed that a temperature shift does not influence the wtcφ105-operators' binding affinity, while the binding of mtcφ105 to the operators was temperature sensitive. This explains how thermo-induction triggers the release of the mutant repressor and renders heterologous gene expression. Interestingly, mtcφ105 and ORF4 demonstrated a large affinity discrepancy towards individual operators at different temperatures. mRNA levels monitored by real-time RT-PCR indicated a suppression of mtcφ105 expression, but a stimulation of ORF4 transcription after thermo-induction. Our data suggested that ORF4 might be a counter protein to the phage repressor in the modulation of the two divergent-oriented promoters P M and P R within the immF region. © 2003 Elsevier Ltd. All rights reserved.en_HK
dc.languageengen_HK
dc.publisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/jmben_HK
dc.relation.ispartofJournal of Molecular Biologyen_HK
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subjectBacillus subtilisen_HK
dc.subjectBacteriophage φ105en_HK
dc.subjectcts-52 repressoren_HK
dc.subjectReverse transcriptase polymerase chain reactionen_HK
dc.subjectSurface plasmon resonanceen_HK
dc.subject.meshAmino Acid Sequenceen_HK
dc.subject.meshBacillus - virologyen_HK
dc.subject.meshBacillus Phages - genetics/metabolismen_HK
dc.subject.meshGene Expression Regulation, Viralen_HK
dc.subject.meshSequence Analysis, DNAen_HK
dc.titleInteraction of a putative transcriptional regulatory protein and the thermo-inducible cts-52 mutant repressor in the Bacillus subtilis phage φ105 genomeen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0022-2836&volume=333&issue=1&spage=21&epage=31&date=2003&atitle=Interaction+of+a+putative+transcriptional+regulatory+protein+and+the+thermo-inducible+cts-52+mutant+repressor+in+the+Bacillus+subtilis+phage+phi105+genomeen_HK
dc.identifier.emailLim, BL: bllim@hkucc.hku.hken_HK
dc.identifier.authorityLim, BL=rp00744en_HK
dc.description.naturepostprinten_HK
dc.identifier.doi10.1016/j.jmb.2003.08.017en_HK
dc.identifier.pmid14516740-
dc.identifier.scopuseid_2-s2.0-0141792426en_HK
dc.identifier.hkuros85497-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0141792426&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume333en_HK
dc.identifier.issue1en_HK
dc.identifier.spage21en_HK
dc.identifier.epage31en_HK
dc.identifier.isiWOS:000185794700002-
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.scopusauthoridChan, AY=26021082600en_HK
dc.identifier.scopusauthoridLim, BL=7201983917en_HK

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