ABSTRACT
The controller protein of the type II restriction-modification (RM) system Esp1396I binds to three distinct DNA operator sequences upstream of the methyltransferase and endonuclease genes in order to regulate their expression. Previous biophysical and crystallographic studies have shown molecular details of how the controller protein binds to the operator sites with very different affinities. Here, two protein-DNA co-crystal structures containing portions of unbound DNA from native operator sites are reported. The DNA in both complexes shows significant distortion in the region between the conserved symmetric sequences, similar to that of a DNA duplex when bound by the controller protein (C-protein), indicating that the naked DNA has an intrinsic tendency to bend when not bound to the C-protein. Moreover, the width of the major groove of the DNA adjacent to a bound C-protein dimer is observed to be significantly increased, supporting the idea that this DNA distortion contributes to the substantial cooperativity found when a second C-protein dimer binds to the operator to form the tetrameric repression complex.
Subject(s)
Bacterial Proteins/chemistry , DNA Restriction-Modification Enzymes/chemistry , DNA, Bacterial/chemistry , DNA-Binding Proteins/chemistry , Escherichia coli/chemistry , Gene Expression Regulation, Bacterial , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Crystallography, X-Ray , DNA Restriction-Modification Enzymes/genetics , DNA, Bacterial/genetics , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Operator Regions, Genetic/genetics , Protein Conformation , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Modification of proteins by methylation has emerged as a key regulatory mechanism in many cellular processes, including gene control. Eighty to ninety percent of the arginine methylation in the cell is performed by the protein arginine methyl transferase PRMT1. ILF3, a protein involved in gene regulation at several levels, has been shown to be a substrate and regulator of PRMT1 in mammals. Here we show that the Xenopus orthologue of ILF3 (Xilf3) is methylated in vivo, and, at least in vitro, this methylation is carried out by Xprmt1b. The in vitro methylation of Xilf3 inhibits its ability to bind to DNA while leaving RNA binding activity unaltered. Consistent with these activities having a role in vivo, the DNA binding activity of the Xilf3-containing CBTF complex and the transcription of its target gene, Xgata2, are both decreased by overexpression of Xprmt1b in embryos. However, in contrast to other RNA binding proteins, a changing degree of methylation does not alter the subcellular localization of Xilf3. Several other proteins involved in gene regulation can bind both RNA and DNA; these data demonstrate a mechanism by which such binding activities may be controlled independently.
Subject(s)
DNA/metabolism , Methyltransferases/metabolism , Nuclear Factor 90 Proteins/metabolism , RNA-Binding Proteins/metabolism , RNA/metabolism , Xenopus Proteins/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , DNA/genetics , DNA Methylation , Methyltransferases/genetics , Molecular Sequence Data , Nuclear Factor 90 Proteins/genetics , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA/genetics , Xenopus , Xenopus Proteins/geneticsABSTRACT
Controller (C) proteins regulate the timing of the expression of restriction and modification (R-M) genes through a combination of positive and negative feedback circuits. A single dimer bound to the operator switches on transcription of the C-gene and the endonuclease gene; at higher concentrations, a second dimer bound adjacently switches off these genes. Here we report the first structure of a C protein-DNA operator complex, consisting of two C protein dimers bound to the native 35 bp operator sequence of the R-M system Esp1396I. The structure reveals a role for both direct and indirect DNA sequence recognition. The structure of the DNA in the complex is highly distorted, with severe compression of the minor groove resulting in a 50 degrees bend within each operator site, together with a large expansion of the major groove in the centre of the DNA sequence. Cooperative binding between dimers governs the concentration-dependent activation-repression switch and arises, in part, from the interaction of Glu25 and Arg35 side chains at the dimer-dimer interface. Competition between Arg35 and an equivalent residue of the sigma(70) subunit of RNA polymerase for the Glu25 site underpins the switch from activation to repression of the endonuclease gene.
