Thermodynamic and enzymological characterization of the interaction between transcription termination factor rho and lambda cro mRNA.
Termination of transcription at tR1, the rho-dependent terminator between genes cro and cII of bacteriophage lambda, is mediated by interactions between rho protein and an RNA sequence element called rut. We show, using a filter retention assay technique, that rho protein binds with about 10-fold lower affinity to variants of cro RNA lacking both parts of rut or to normal cro RNA having one or the other part of rut bound to a complementary DNA oligonucleotide than it binds to unmodified cro RNA. These same variant and modified forms are nearly devoid of the strong rho ATPase cofactor activity of cro RNA. Estimates of binding energies of the rho-cro RNA interaction under different conditions reveal that termination function correlates with about 12.6 kcal of binding energy, of which two-thirds is due to nonelectrostatic interactions. The rut segment is shown to contribute about 1 kcal, nearly all to nonelectrostatic interactions. KCl is found to be more effective than potassium glutamate as a competitive counterion, and a decrease in 1.4 kcal of binding energy due to counterion competition correlates with a loss of termination and ATPase activities. In sum, the results indicate that the rut sequence contributes substantially to the overall binding affinity, that ionic interactions are also important, and that mere binding of rho to RNA is not sufficient for rho ATPase activation.
Faus
I
I
Department of Chemistry, Indiana University, Bloomington 47405.
Richardson
J P
JP
D000251:Adenosine Triphosphatases / Q000378:metabolism; D010582:Bacteriophage lambda / Q000378:metabolism; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D012234:Rho Factor / Q000378:metabolism*; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism*
D003067:Coenzymes; D012333:RNA, Messenger; D012367:RNA, Viral; D012234:Rho Factor; D014157:Transcription Factors; D000251:Adenosine Triphosphatases