Chan Annie Y, Lim Boon L
Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong, People's Republic of China.
J Mol Biol. 2003 Oct 10;333(1):21-31. doi: 10.1016/j.jmb.2003.08.017.
A 144 amino acid residue cts-52 mutant repressor (mtc phi 105) located in the EcoRI-F immunity region (immF) of Bacillus subtilis phage phi 105 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 alpha helix-beta turn-alpha 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 (wtc phi 105) and the mutant repressor mtc phi 105, 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 wtc phi 105-operators' binding affinity, while the binding of mtc phi 105 to the operators was temperature sensitive. This explains how thermo-induction triggers the release of the mutant repressor and renders heterologous gene expression. Interestingly, mtc phi 105 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 mtc phi 105 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.
位于枯草芽孢杆菌噬菌体φ105的EcoRI - F免疫区域（immF）中的一个144个氨基酸残基的cts - 52突变阻遏物（mtc φ105）参与了一个热诱导表达系统的调控机制。在阻遏物基因旁边，一个名为ORF4的开放阅读框编码一个90个氨基酸残基的多肽，该多肽与阻遏物的氨基酸序列具有37%的同源性。基于蛋白质序列比对，在阻遏物的N端区域（第18 - 37位残基）以及ORF4的多肽（第22 - 41位残基）中鉴定出一个DNA结合α螺旋 - β转角 - α螺旋（HTH）基序。通过实时逆转录聚合酶链反应（RT - PCR）和蛋白质免疫印迹分析证实了突变阻遏物和ORF4在体内的表达。为了研究它们的DNA结合特性，野生型阻遏物（wtc φ105）和具有苏氨酸17到异亮氨酸替换的突变阻遏物mtc φ105在大肠杆菌中过表达并纯化用于亲和测定。通过表面等离子体共振（SPR）阐明了它们在不同温度下对六个操纵位点的亲和力。我们的数据表明温度变化不影响wtc φ105与操纵位点的结合亲和力，而mtc φ105与操纵位点的结合对温度敏感。这解释了热诱导如何触发突变阻遏物的释放并实现异源基因表达。有趣的是，mtc φ105和ORF4在不同温度下对各个操纵位点表现出很大的亲和力差异。通过实时RT - PCR监测的mRNA水平表明热诱导后mtc φ105的表达受到抑制，但ORF4的转录受到刺激。我们的数据表明，在调节immF区域内两个方向相反的启动子P(M)和P(R)时，ORF4可能是噬菌体阻遏物的一种拮抗蛋白。
