Identification and characterization of the DNA-binding domain of the multifunctional PutA flavoenzyme
Identification and characterization of the DNA-binding domain of the multifunctional PutA flavoenzyme
复制标题
DOI：
10.1074/jbc.m403701200
复制
发表时间：
2004-07-23
影响因子：
4.8
通讯作者：
Becker, DF
中科院分区：
文献类型：
--
作者：
Gu, D;Zhou, YZ;Becker, DF
大肠杆菌的PutA黄素蛋白是一种转录抑制因子，也是一种调节和催化脯氨酸氧化的双功能酶。PutA通过与put调节子的控制DNA区域结合来抑制基因putA和putP的转录。本研究的目的是确定和表征PutA的DNA结合结构域。PutA是一种由1320个氨基酸组成的多肽，其DNA结合活性定位于N-末端残基1 - 261。在探索了PutA的潜在DNA结合区域和N端缺失突变体后，确定残基1 - 90（PutA 90）含有DNA结合活性并稳定PutA的二聚体结构。基于细胞的转录测定证明PutA 90在体内作为转录阻遏物起作用。PutA 90与put对照DNA的解离常数估计为110 nM，略高于PutA-DNA复合物的解离常数（K-d类似于45 nM）。PutA 90的一级和二级结构分析表明，在残基1 - 47中存在带-螺旋-螺旋DNA结合基序。为了验证这一预测，我们纯化并表征了PutA 47。PutA 47显示纯化为明显的二聚体，表现出体内转录活性，并特异性结合到放对照DNA。在凝胶迁移率变动分析中，观察到PutA 47与放置对照DNA协同结合，PutA 47-DNA复合物的总解离常数为15 nM。因此，N-末端残基1 - 47对于DNA结合和PutA的二聚体结构至关重要。这些结果与转录因子的带-螺旋-螺旋家族一致。
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 similar to 45 nM). Primary and secondary structure analysis of PutA90 suggested the presence of a ribbon-helix-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, N-terminal 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.