Escherichia coli transcription termination factor ρ has a two-domain structure in its activated form

Limited tryptic digestion of Escherichia coli transcription termination factor ρ [an RNA-dependent nucleoside triphosphatase (NTPase)] yields predominantly two fragments (f1 and f2) when the protein is bound to both poly(C) and ATP. The apparent molecular masses of the two fragments are 31 kDa for f1 and 15 kDa for f2, adding up to the molecular mass of the intact ρ polypeptide chain (46 kDa). Sequence analysis of the amino termini demonstrates that f1 is derived from the amino-terminal portion of ρ and that the trypsin cleavage that defines f2 occurs at lysine-283. These results suggest that, in the liganded (activated) form, the native ρ protein monomer is organized into two distinct structural domains that are separable by a single proteolytic cleavage. The f1 fragment, purified from NaDodSO₄/polyacrylamide gels and renatured, binds poly(C) but the f2 fragment does not; neither regains any ATPase activity. ATP- and polynucleotide-dependent changes in the rate of proteolysis and in the character of the fragments produced suggest that ρ undergoes a series of conformational transitions as a consequence of RNA binding, NTP binding and NTP hydrolysis. The rate of loss of ρ ATPase activity and of intact ρ monomers is slower in the presence of adenosine 5'-[γ-thio]triphosphate than in the presence of either ATP or ADP, indicating that the hydrolysis of ATP may result in different conformational effects than does the binding of this ligand. These findings are discussed within the context of recent models of ρ-dependent transcription termination.