The rho-115 mutation in transcription termination factor rho affects its primary polynucleotide binding site.
We have investigated the effect of the rho-115 mutation on the catalytic properties of the Escherichia coli termination protein, rho. Comparison of the primary and secondary polynucleotide binding sites activities reveals dramatic differences between the mutant and wild-type molecules. Wild-type rho must bind single-stranded polynucleotides to activate its nucleotide triphosphatase (NTPase) activity, and either poly(C), or poly(dC) plus oligo(C), will suffice. In contrast, attempted activation of the rho-115 NTPase with poly(C) in the presence of poly(dC) showed the latter to be a potent inhibitor. Inclusion of small oligonucleotides such as oligo(C) in the activation assay does not inhibit the poly(C)-induced NTPase reaction of either wild-type rho or rho-115. This would indicate, in the two polynucleotide binding site model for rho proposed by Richardson (Richardson, J.P. (1982) J. Biol. Chem. 251, 5760-5766), that the mutation in rho-115 affects the primary polynucleotide binding site. Transcription termination in vitro at the rho-dependent site trp t' showed dramatically reduced termination with rho-115 protein compared to wild-type rho. In the presence of rho-115, the transcript is longer and termination occurs over a narrower range of nucleotides than with wild-type rho. This suggests that the primary polynucleotide binding site is important not only for efficient termination of transcription but may also be involved in determining the terminal end point of the transcript itself.
D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009154:Mutation; D043583:Nucleoside-Triphosphatase; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011066:Poly C / Q000378:metabolism; D011119:Polynucleotides / Q000378:metabolism*; D012234:Rho Factor / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D011119:Polynucleotides; D012234:Rho Factor; D014157:Transcription Factors; C033539:oligodeoxycytidylic acid; C023826:poly(dC); D011066:Poly C; D010744:Phosphoric Monoester Hydrolases; D043583:Nucleoside-Triphosphatase