Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity

Abstract

Repressors encoded by mycobacteriophages differ significantly from the repressor proteins of lambda and related phages at functional level but at structural level they are nearly similar.

Antagonistic effects Na+ and Mg2+ on the structure, function, and stability of mycobacteriophage L1 repressor

“…The CD spectrum (200-260 nm) of NaCl-equilibrated CTD contained a peak of large negative ellipticity at 208 nm, while the corresponding peak from the CTD spectrum in MgCl2 buffer was substantially reduced . As expected, a significant reduction of α-helical and an increase of random coil structure in CTD occurred when NaCl was replaced with MgCl2, indicating that Na ＋ was superior to Mg 2＋ at maintaining the conformation of CTD, the most stable region of CI (8).…”

“…Recently, CI was purified to homogeneity and characterized to some extent (8). The requirement of Na ＋ for maintaining the biologically active conformation of CI was investigated by equilibration of CI in NaCl, KCl, LiCl, or NH4Cl buffer and the structure and function of the CI examined by standard in vitro methods and comparison of the results.…”

“…The CTD of L1 repressor, which carries six Trp residues, has been reported to be resistant to chymotrypsin digestion (8), indicating that all the six Trp residues might be buried in the folded CTD interior and that the intrinsic fluorescence from such inaccessible Trp residues should display a blue-shifted emission maximum. Tryptophan fluorescence spectrum of CTD in NaCl buffer, in fact, showed an emission maximum at 335.6 nm (Fig.…”

“…Purification of histidine-tagged L1 repressor (CI) was described previously (8). The C-terminal domain of L1 repressor (CTD) was purified by partial digestion of repressor with chymotrypsin followed by the purification of CTD as previously described for purification of trypsin-generated CTD (8).…”

“…L1 CI is a 183 residue polypeptide with a small hinge region flanked by an N-terminal domain (NTD) and a C-terminal domain (CTD). Both CTD and CI form dimers in solution, have a substantial amount of α-helix at 30 o C, and unfold at higher temperatures (8). L1 CI binds to different operator DNAs with variable affinity in a temperature sensitive manner.…”