GENE CLONING, EXPRESSION, AND CHARACTERIZATION OF THE SAC7 PROTEINS FROM THE HYPERTHERMOPHILE SULFOLOBUS-ACIDOCALDARIUS

The genes for two Sac7 DNA-binding proteins, Sac7d and Sac7e, from the extremely thermophilic archaeon SulSolobus acidocaldarius have been c loned into Escherichia coil and sequenced. The sac7d and sac7e open re ading frames encode 66 amino acid (7608 Da) and 65 amino acid (7469 Da ) proteins, respectively. Southern blots indicate that these are the o nly two Sac7 protein genes in S. acidocaldarius, each present as a sin gle copy. Sac7a, b: and c protein appear to be carboxy-terminal modifi ed Sac7d species. The transcription initiation and termination regions of the sac7d and sac7e genes have been identified,along with the prom oter elements. Potential ribosome binding sites have been identified d ownstream of the initiator codons. The sac7d gene has been expressed i n E. coli, and various physical properties of the recombinant protein have been compared with those of native Sac7. The UV absorbance spectr a and extinction coefficients, the fluorescence excitation and emissio n spectra, the circular dichroism, and the two-dimensional double-quan tum filtered H-1 NMR spectra of the native and recombinant species are essentially identical, indicating essentially identical local and glo bal folds. The recombinant and native proteins bind and stabilize doub le-stranded DNA with a site size of 3.5 base pairs and an intrinsic bi nding constant of 2 x 10(7) M(-1) for poly[dGdC]. poly[dGdC] in 0.01 M KH2PO4 at pH 7.0. The availability of the recombinant protein permits a direct comparison of the thermal stabilities of the methylated and unmethylated forms of the protein. Differential scanning calorimetry d emonstrates that the native protein is extremely thermostable and unfo lds reversibly at pH 6.0 with a T-m, of approximately 100 degrees C, w hile the recombinant protein unfolds at 92.7 degrees C.