Cloning, expression, secondary structure characterization of HMG box 1 of hUBF from E. coli and its binding to DNA.
Human upstream binding factor (hUBF) belonging to a family of protein containing DNA binding domain-HMG box, is important in the activation of rRNA gene transcription. It contains six tandemly arranged HMG box domains, each of which is thought to be as a basic architectural unit in the interaction of DNA and protein. Here the DNA binding domain of hUBF HMG box 1 was cloned and heterologously expressed in Escherichia coli. Through a single purification step using a Ni2+-chelating column, the highly purified recombinant protein could be obtained. This recombinant protein contains 99 amino acids with a hexahistidine tag added to the C-terminus. It was expressed as a monomer, which was determined by gel filtration. Circular dischroism studies show that it comprises approximately 54.3% alpha-helix and 43.6% random coil at pH 7. This result is in good agreement with that of FTIR, which are 59.9% alpha-helix and 40.1% random coil. There is no obvious change for the secondary structure of the recombinant protein as increasing pH from 5.0 to 12.0. But denaturation occurs at pH 3.0. Like many HMG box domains that were found in other proteins, it could bind to four-way DNA junction, a putative intermediate in DNA recombination, in a structure-specific manner. Magnesium ion has no effect on this binding activity, which is determined by both gel mobility shift assays and surface plasmon resonance (SPR). Since Mg2+ is present in the nucleus and RNA polymerase I is Mg2+-stimulated, we believe that this property is relevant for hUBF in vivo. SPR research shows that the recombinant hUBF HMG box 1 also has a strong binding ability to a GC-rich fragment within the rRNA gene core promoter.
Yang
Wulin
W
Laboratory of Structure Biology, School of Life Science, University of Science and Technology of China, Anhui, Hefei, People's Republic of China.
Zeng
Wanyong
W
Zhou
Dan
D
Shi
Yunyu
Y
D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D035463:Pol1 Transcription Initiation Complex Proteins; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D012335:RNA, Ribosomal / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; D012335:RNA, Ribosomal; D011994:Recombinant Proteins; D014157:Transcription Factors; C063844:transcription factor UBF; D004247:DNA