Specificity and efficiency of rho-factor helicase activity depends on magnesium concentration and energy coupling to NTP hydrolysis.
The RNA-DNA helicase activity of Escherichia coli transcription termination factor rho can be significantly enhanced at lower potassium chloride and magnesium acetate concentrations than previously used. Decreasing the potassium chloride concentration from 150 to 50 mM increases the rate of release at least 4-fold, while at lower magnesium concentrations less ATP is required for maximal duplex disruption. For all concentrations tested (between 0.1 and 5 mM), the optimal magnesium and ATP concentrations are interdependent; a roughly equimolar ratio gives the maximal rate of RNA release, although peak height and breadth vary. Surprisingly, rho behaves differently with an RNA-RNA duplex, which cannot be efficiently disrupted at magnesium concentrations below 1 mM. Above 2.0 mM, release does occur efficiently suggesting that Mg2+ promotes some structural transition in the RNA-RNA helix to a rho-susceptible conformation. In addition to Mg2+, helicase activity requires hydrolysis of nucleoside triphosphates, but for all four standard NTPs the rates of NTP hydrolysis do not correlate uniformly with the rates of RNA release. Based on the ratio of the rate of RNA release to the rate of NTP hydrolysis, rho utilizes ATP most efficiently. The 2-4-fold weaker coupling of hydrolysis to duplex disruption for the other three NTPs demonstrates that NTP utilization is not, on its own, sufficient for efficient helicase activity. The less efficient coupling with GTP, CTP, and UTP correlates with conformational differences in the protein complex as probed by mild trypsin digestion. The implications of our findings for substrate specificity and energy coupling in the helicase reaction are discussed.
Brennan
C A
CA
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Steinmetz
E J
EJ
Spear
P
P
Platt
T
T
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003570:Cytidine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004734:Energy Metabolism; D004926:Escherichia coli / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D012234:Rho Factor / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000378:metabolism
D009711:Nucleotides; D012234:Rho Factor; D014157:Transcription Factors; D012313:RNA; D003570:Cytidine Triphosphate; D011189:Potassium Chloride; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C031715:rho-ATPase; D008274:Magnesium; D014544:Uridine Triphosphate