No abstract

The crystal structure of the complex between Eco RI endonuclease and the cognate oligonucleotide TCGCGAATTCGCG provides a detailed example of the structural basis of sequence-specific DNA-protein interactions. The structure was determined, to 3 A resolution, by the ISIR (iterative single isomorphous replacement) method with a platinum isomorphous derivative. The complex has twofold symmetry. Each subunit of the endonuclease is organized into an alpha/beta domain consisting a five-stranded beta sheet, alpha helices, and an extension, called the "arm," which wraps around the DNA. The large beta sheet consists of antiparallel and parallel motifs that form the foundations for the loops and alpha helices responsible for DNA strand scission and sequence-specific recognition, respectively. The DNA cleavage site is located in a cleft that binds the DNA backbone in the vicinity of the scissile bond. Sequence specificity is mediated by 12 hydrogen bonds originating from alpha helical recognition modules. Arg200 forms two hydrogen bonds with guanine while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. These interactions discriminate the Eco RI hexanucleotide GAATTC from all other hexanucleotides because any base substitution would require rupture of at least one of these hydrogen bonds.

The original model of Eco RI endonuclease (1) proved refractory to crystallographic refinement, whereby model coordinates were adjusted to optimize the fit to the observed diffraction data (the best R factor for the original model is 0.25). We The new data yielded a multiple isomorphous replacement (MIR) electron density map that suggests a different connectivity between elements of secondary structure in the protein (3). A model based on that connectivity has refined robustly by the X-PLOR and Konnert-Hendrickson methods (4) to an R factor of 0.20 (no ordered solvent has been included in this model, nor have coordinates for the first 16 amino acid residues, which appear disordered) (5). The root-mean-square deviations in bond distance, angle distance, and 1-4 dihedral distance are 0.016 A, 0.031 A, and 0.031 A,

The substrate specificity of the EcoRI restriction endonuclease can be varied in vitro by changing the pH and the ionic environment of the reaction. Phosphodiester bond cleavage occurs at a DNA hexanucleotide sequence

The complete nucleotide sequences of the Haemophilus influenzae and Mycoplasma genitalium genomes and the partially sequenced Escherichia coli chromosome were analyzed to identify open reading frames (ORFs) likely to encode RNA modifying enzymes. The protein sequences of known RNA modifying enzymes from three families--m5U methyltransferases, psi synthases and 2'-O methyltransferases--were used as probes to search sequence databases for homologs. ORFs identified as homologous to the initial probes were retrieved and used as new probes against the databases in an iterative manner until no more homologous ORFs could be identified. Using this approach, we have identified two new m5U methyltransferases, seven new psi synthases and four new 2'-O methyltransferases in E. coli. Many of the ORFs found in E.coli have direct genetic counterparts (orthologs) in one or both of H.influenzae and M.genitalium. Since there is a near-complete knowledge of RNA modifications in E.coli, functional activities of the proteins encoded by the identified ORFs were proposed based on the level of conservation of the ORFs and the modified nucleotides.