Temperature-sensitive mutant rho-115 rho-RNA binary complexes, and stabilization by substrates and analogues.
To determine the molecular basis for the temperature-sensitivity of pure rho RNA-dependent ATPase from Escherichia coli mutant rho-115 cells, we investigated mutant rho binding to [3H] polyC as measured by retention on nitrocellulose filters. Complexes of wild-type rho and polyC incubated at 37 degrees C and 45 degrees C were similarly stable. At 37 degrees C mutant rho-polyC binary complexes were inactivated at a slightly faster rate than complexes with wild-type rho. Upon shift to 45 degrees C the quantity of rho-115 bound to polyC declined immediately, resulting in one-fifth of the quantity of complexes observed at 37 degrees C. Shift back to 37 degrees C restored the level of observed complexes by two-fold. The inclusion of ATP or the analogue beta-gamma methylene ATP during 45 degrees C incubation resulted in stable mutant rho-polyC complexes. The hydrolysis product ADP was also effective in stabilizing binary complexes at 45 degrees C but this effect was observed with an order of magnitude more ADP than ATP. Adenine, adenosine, AMP or Pi had no stabilizing effect. We conclude that the mutant rho-115 protein exhibits a structural instability as a result of binding RNA. Furthermore ATP confers a wild-type phenotype upon rho-115 protein, probably as a result of conformational change due to binding of this compound. The effect of ATP on the stability of mutant rho-polyC binary complexes supports the model of ATP modulation of rho-RNA interaction proposed by Galluppi and Richardson (1980).
D000225:Adenine / Q000378:metabolism; D000241:Adenosine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D009154:Mutation; D011066:Poly C / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012234:Rho Factor / Q000378:metabolism*; D013696:Temperature; D014157:Transcription Factors / Q000378:metabolism*
D012333:RNA, Messenger; D012234:Rho Factor; D014157:Transcription Factors; D011066:Poly C; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000225:Adenine; D000241:Adenosine