In vitro Identification and Characterization of DNA Sequences Bound by Brevibacillus thermoruber WR-249 Lon Protease
Date Issued
2005
Date
2005
Author(s)
Lee, Huai-Cheng
DOI
en-US
Abstract
Proteins with DNA-binding activity mostly play an important role in the gene expression of a cell. This kind of proteins appears to exert its regulatory function by degrading some transcription factors after it binds to DNA. To investigate what kinds of elements bound by the DNA-binding proteins, identification of the binding site of the DNA must be achieved.
A thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized with an N-terminal domain, a central ATPase domain including a SSD (sensor- and substrate- discrimination) domain, and a C-terminal protease domain. Bt-Lon is a multi-functional enzyme and its functions include the degradation of proteins, ATPase, chaperone-like activities, and DNA binding.
Gel mobility shift assays revealed that Bt-Lon binds to DNA cooperatively. In line with the previous result, we demonstrated that SSD domain is involved in the DNA-binding activity by recruiting a truncated mutant, Bt-Lon-C289. It was also found that Bt-Lon, as well as the E.coli and human Lon, exhibits a high affinity to the TG-rich DNA. To further investigate whether Bt-Lon binds DNA specifically, DNA fragments bound by Bt-Lon were purified and then subjected to MS analysis and sequence determination by cloning/sequencing on the vector (pCR4Blunt-TOPO).
Experimental data revealed that the five fragments bound by Bt-Lon from plasmid pET28a were homologous in sequence with the elements bound by E. coli and human Lon. Furthermore, we selected and cloned the five fragments, encompassing ms1, ms2, lb1, lb2 and lb3 by PCR amplification. Based on the high affinity of these five fragments to Bt-Lon, it is suggested that Lon binds to DNA dependent on the site-specific TG-rich DNA sequences, and the secondary or tertiary structure of the plasmid DNA can enhance the DNA-binding affinity of Bt-Lon.
A thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized with an N-terminal domain, a central ATPase domain including a SSD (sensor- and substrate- discrimination) domain, and a C-terminal protease domain. Bt-Lon is a multi-functional enzyme and its functions include the degradation of proteins, ATPase, chaperone-like activities, and DNA binding.
Gel mobility shift assays revealed that Bt-Lon binds to DNA cooperatively. In line with the previous result, we demonstrated that SSD domain is involved in the DNA-binding activity by recruiting a truncated mutant, Bt-Lon-C289. It was also found that Bt-Lon, as well as the E.coli and human Lon, exhibits a high affinity to the TG-rich DNA. To further investigate whether Bt-Lon binds DNA specifically, DNA fragments bound by Bt-Lon were purified and then subjected to MS analysis and sequence determination by cloning/sequencing on the vector (pCR4Blunt-TOPO).
Experimental data revealed that the five fragments bound by Bt-Lon from plasmid pET28a were homologous in sequence with the elements bound by E. coli and human Lon. Furthermore, we selected and cloned the five fragments, encompassing ms1, ms2, lb1, lb2 and lb3 by PCR amplification. Based on the high affinity of these five fragments to Bt-Lon, it is suggested that Lon binds to DNA dependent on the site-specific TG-rich DNA sequences, and the secondary or tertiary structure of the plasmid DNA can enhance the DNA-binding affinity of Bt-Lon.
Subjects
蛋白結合之DNA序列
DNA結合蛋白
Lon蛋白酶
DNA sequences bound by protein
DNA-binding protein
Lon protease
Type
other