RECONSTITUTION OF ESCHERICHIA-COLI RNASE HI FROM THE N-FRAGMENT WITH HIGH HELICITY AND THE C-FRAGMENT WITH A DISORDERED STRUCTURE

The Escherichia coli RNase HI variant with the Lys(86) --> Ala mutatio n is purified in two forms, as nicked and intact proteins. The nicked K86A protein, in which the N-fragment (Met(1)-Lys(87)) and the C-fragm ent (Arg(88)- Val(155)) remain associated, is enzymatically active. Th ese N- and C-fragments were isolated and examined for reassociation. T hese peptides did not associate to form the nicked K86A protein at pH 3.0 in the absence of salt, but were associated, with a yield of 30-80 %, when the pH was raised to 5.5 or when salt was added. Measurements of the CD spectra show that the alpha-helices are partially formed in the N-fragment at pH 3.0 in the absence of salt and are almost fully f ormed either at pH 5.5 or at pH 3.0 in the presence of 0.15 M NaCl. In contrast, the C-fragment remains almost fully disordered under these conditions. The N-fragment with this high (native-like) helicity shows the characteristics of a molten globule with respect to the content o f the secondary and tertiary structures, the ability to bind a fluores cent probe (1-anilinonaphthalene-8-sulfonic acid), and the behavior on the thermal transition. These results suggest that the N-fragment con tains an initial folding site, probably the alpha I-helix, and the com pletion of the folding in this site provides a surface that facilitate s the folding of the C-fragment. This folding process may represent th at of the intact RNase HI molecule.