ABSTRACT
Two site-specific DNA methyltransferases, M.BcoKIA and M.BcoKIB, were isolated from the thermophilic strain Bacillus coagulans K. Each of the methylases protects the recognition site 5'-CTCTTC-3'/5'-GAAGAG-3' from cleavage with the cognate restriction endonuclease BcoKI. It is shown that M.BcoKIB is an N6-adenine specific methylase and M.BcoKIA is an N4-cytosine specific methylase. According to bisulfite mapping, M.BcoKIA methylates the first cytosine in the sequence 5'-CTCTTC-3'.
Subject(s)
Adenine/chemistry , Bacillus/enzymology , Cytosine/chemistry , DNA Modification Methylases/chemistry , DNA Modification Methylases/isolation & purification , DNA-Cytosine Methylases/chemistry , DNA-Cytosine Methylases/isolation & purification , Deoxyribonucleases, Type II Site-Specific/chemistry , Site-Specific DNA-Methyltransferase (Adenine-Specific)/chemistry , Site-Specific DNA-Methyltransferase (Adenine-Specific)/isolation & purification , Chromatography, Liquid , DNA Methylation , Substrate SpecificityABSTRACT
Thermostable RNA-binding protein Hfq (also denoted HF1) is a multifunctional expression regulator of many bacterial genes. The regulation takes place both at a translation level (directly) and transcription level (indirectly through the stimulation of bacterial RNA polymerase sigmaS-subunit translation). We have cloned and overexpressed the hfq gene from E. coli and developed a purification procedure for the protein. Using gel filtration and ultracentrifugation techniques it was shown that the obtained Hfq protein is highly homogeneous and well dissolved. It has been crystallized and can be used for structural investigations.
Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Escherichia coli/genetics , Escherichia coli/metabolism , Host Factor 1 Protein/genetics , Host Factor 1 Protein/isolation & purification , Amino Acid Sequence , Chromatography, Gel/methods , Cloning, Molecular , Crystallization , DNA Primers/genetics , Escherichia coli/chemistry , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/chemistry , Gene Expression Regulation, Bacterial , Genes, Bacterial/genetics , Host Factor 1 Protein/biosynthesis , Host Factor 1 Protein/chemistry , Molecular Sequence Data , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/isolation & purification , Sequence Homology, Amino Acid , Ultracentrifugation/methodsABSTRACT
We recently isolated a site-specific adenine DNA-methylase, M.BspST5I, which methylates only one strand of the recognized site GCATC. The methylated base is indicated by an asterisk.