Stabilizing interactions in the dimer interface of alpha-subunit in Escherichia coli RNA polymerase: A graph spectral and point mutation study

The formation of alpha (2) dimer in Escherichia coli core RNA polymerase (R NAP) is thought to be the first step toward the assembly of the functional enzyme. A large number of evidences indicate that the alpha -subunit dimeri zes through its N-terminal domain (NTD). The crystal structures of the cl-s ubunit NTD and that of a homologous Thermus aquaticus core RNAP are known. To identify the stabilizing interactions in the dimer interface of the alph a -NTD of E. coli RNAP, we identified side-chain clusters by using the crys tal structure coordinates of E. coli alpha -NTD. A graph spectral algorithm was used to identify side-chain clusters. This algorithm considers the glo bal nonbonded side-chain interactions of the residues for the clustering pr ocedure and is unique in identifying residues that make the largest number of interactions among the residues that form clusters in a very quantitativ e way. By using this algorithm, a nine-residue cluster consisting of polar and hydrophobic residues was identified in the subunit interface adjacent t o the hydrophobic core. The residues forming the cluster are relatively rig id regions of the interface, as measured by the thermal factors of the resi dues. Most of the cluster residues in the E. coli enzyme were topologically and sequentially conserved in the T. aquaticus RNAP crystal structure. Res idues 35F and 46I were predicted to be important in the stability of the al pha -dimer interface, with 35F forming the center of the cluster. The predi ctions were tested by isolating single-point mutants alpha -F35A and alpha -I46S on the dimer interface, which were found to disrupt dimerization. Thu s, the identified cluster at the edge of the dimer interface seems to be a vital component in stabilizing the alpha -NTD.