THE MAJOR DIMERIZATION DETERMINANTS OF THE NITROGEN REGULATORY PROTEIN NTRC FROM ENTERIC BACTERIA LIE IN ITS CARBOXY-TERMINAL DOMAIN

The NTRC protein (nitrogen regulatory protein C) of enteric bacteria i s an enhancer-binding protein that activates transcription by the sigm a(54)-holoenzyme form of RNA polymerase. NTRC is a homodimeric protein that binds to a dyad-symmetrical site in DNA. To activate transcripti on NTRC must be phosphorylated and must form an appropriate oligomeric species at an enhancer. In order to study subunit exchange between NT RC dimers, we constructed a fusion of the maltose-binding protein (MBP ) to the amino-terminal end of NTRC (MBP-NTRC) and visualized the form ation of heterodimers between MBP-NTRC and wild-type NTRC by a gel-mob ility shift assay for DNA-binding. When MBP-NTRC is mixed with wild-ty pe NTRC at 37 degrees C, subunit exchange occurs rapidly. The apparent half-life for dissociation of homodimers of NTRC is two to three minu tes at 37 degrees C and is not changed by phosphorylation. The isolate d carboxy-terminal domain of NTRC (91 amino acid residues) forms heter odimers with both wild-type NTRC and MBP-NTRC, indicating that the C-t erminal domain is sufficient for dimerization. The apparent rate of di ssociation of homodimers of the C-terminal domain is essentially the s ame as that of full-length NTRC, indicating that the major dimerizatio n determinants of the protein lie in its C-terminal domain. Congruent with this, a truncated form of NTRC from which the last 58 amino acid residues were removed is a monomer in solution. Moreover, truncated fo rms of NTRC from which the last 16 or 26 amino acid residues were remo ved are predominantly monomeric in solution, as is a mutant form with the amino acid substitution A410E in its C-terminal domain. Monomeriza tion of the above mutant; forms of NTRC can be rationalized on the bas is of homology between the C-terminal region of NTRC and a 50 amino ac id residue region of the factor for inversion stimulation (FIS) protei n.