P J Greene

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 d(N-G-A-A-T-T-C-N)/d(N-C-T-T-A-A-G-N) when the ionic strength is high, 100 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, and the pH is approximately 7.3. Lowering the ionic strength to 25 mM Tris-HCl, 2 mM MgCl2, and adjusting the pH to 8.5 reduces the recognition specificity of the EcoRI endonuclease to the tetranucleotide sequence, d(N-A-A-T-T-N)/d(N-T-T-A-A-N). The enzymatic activity responsible for this substrate recognition is referred to as EcoRI. Cleavage of pVH51 plasmid DNA under EcoRI conditions results in a number of partial digest fragments, some of which disappear slowly over a prolonged digestion period. This suggests that different recognition sites are cleaved at different rates. Comparison of DNA fragment patterns of modified and unmodified pVH51 DNA indicates that the canonical EcoRI sequence is the most rapidly cleaved site under EcoRI conditions. DNA modified in vivo by the EcoRI methylase is not cleaved by the EcoRI endonuclease under standard conditions, but is cleaved under EcoRI conditions at sites other than the standard EcoRI substrate.

The Eco RI endonuclease and methylase recognize the same hexanucleotide substrate sequence. We have determined the sequence of a fragment of DNA which encodes these enzymes using the chain-termination method of Sanger (Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467). The amino acid sequences of both enzymes were derived from the DNA sequence. The coding regions selected include the only open translational frames of sufficient length to accommodate the enzymes. They coincide with previously established gene boundaries and orientation. The predicted amino acid sequences correlate well with analyses of the purified protein. Comparison of the nucleotide and protein sequences reveals no homology between the endonuclease and methylase which might provide insight into the origin of the restriction-modification system or the mechanism of common substrate recognition. Based on secondary structure predictions, the two enzymes also have grossly different molecular architecture. The base composition of the sequence is 65% A + T, and the codon usage is significantly different from that observed in several Escherichia coli chromosomal genes. In some cases, frequently selected codons are recognized by minor tRNA species. A spontaneous mutation in the endonuclease gene was isolated. Serine replaces arginine at residue 187. In crude extracts, Eco RI specific cleavage is approximately 0.3% wild type.

The Escherichia coli fmu gene product has recently been determined to be the 16S rRNA m 5 C 967 methyl-transferase. As such, Fmu represents the first protein identified as an S -adenosyl-L-methionine (AdoMet)-dependent RNA m 5 C methyltransferase whose amino acid sequence is known. Using the amino acid sequence of Fmu as an initial probe in an iterative search of completed DNA sequence databases, 27 homologous ORF products were identified as probable RNA m 5 C methyltransferases. Further analysis of sequences in undeposited genomic sequencing data and EST databases yielded more than 30 additional homologs. These putative RNA m 5 C methyltransferases are grouped into eight subfamilies, some of which are predicted to consist of direct genetic counterparts, or orthologs. The enzymes proposed to be RNA m 5 C methyltransferases have sequence motifs closely related to signature sequences found in the well-studied DNA m 5 C methyltransferases and other AdoMet-dependent methyltransferases. Structure-function correlates in the known AdoMet methyltransferases support the assignment of this family as RNA m 5 C methyltransferases.

The free energy of the binding reaction between EcoRI restriction endonuclease and a specific cognate dodecadeoxynucleotide (d(CGCGAATTCGCG)) has contributions from both electrostatic and nonelectrostatic components. These contributions were dissected by measuring the effects of varying salt concentration on the equilibrium binding constant and applying the thermodynamic analyses of Record et al. (Record, M. T., Jr., Lohman, T. M., and deHaseth, P. L. (1976) J. Mol. Biol. 107, 145-158). Endonuclease mutation S187 (Arg 187 to Ser) (Greene, P. J., Gupta, M., Boyer, H. W., Brown, W. E., and Rosenberg, J. M. (1981) J. Biol. Chem. 256, 2143-2153) did not significantly affect the nonelectrostatic component but did perturb the electrostatic contribution to the binding energy (we are numbering the amino acid residues according to the DNA sequence). The former was determined by extrapolating the linear portion of the salt dependence curve (0.125 to 0.25 M KCl) to 1 M ionic strength, with the same result for both wild type and S187 endonucleases at both pH 6.0 and 7.4 (-8.5 +/- 1.5 kcal/mol or greater than 50% of the total binding free energy). The slopes of these same curves yield estimates of eight ionic interactions between wild type endonuclease and the DNA at both pH values. By contrast, binding of EcoRI-S187 to dodecanucleotide involves six charge-charge interactions at pH 6.0. Only two ionic interactions are observed at pH 7.4. This was unexpected since gel permeation chromatography demonstrated that the recognition complex for both wild type and S187 proteins contains an enzyme dimer and a DNA duplex. EcoRI-S187 endonuclease retains wild type DNA sequence specificity, and the rate of the phosphodiester hydrolysis step is also unchanged. Thus, electrostatic interactions are functionally separable from sequence recognition and strand cleavage. Our results also establish that arginine 187 plays a key role in the electrostatic function and suggest that it might be located at the DNA-protein interface. The disproportionate loss of ion pairs at pH 7.4 can be rationalized by a model which suggests that six conformationally mobile ionic groups on the protein act in a coordinated manner during the interaction with DNA.