Mutations in the ATP-binding domain of Escherichia coli rho factor affect transcription termination in vivo.
Five mutant rho proteins, representing alterations at three different locations in the Escherichia coli rho gene that affect ATP hydrolytic activity but not RNA binding, were examined in vivo for function at the rho-dependent IS2 and bacteriophage lambda tR1 terminators. The altered amino acids in rho are located at highly conserved residues near the beta 1 and beta 4 strands of the hydrophobic ATP-binding pocket that is structurally similar to the F1-type ATPases and adenylate kinase. The RNA-dependent ATPase activities of the mutant rho proteins were previously shown to range from undetectable to a twofold increase over wild-type rho in vitro. Analysis of these proteins within the environment of the cell confirmed that transcription termination in vivo is indeed related to the ability of rho factor to properly hydrolyze nucleoside triphosphates, as would be predicted from results in vitro. The relative efficiency of termination at lambda tR1, as judged by lambda N= plating efficiency and by suppression of polarity of IS2 upstream of galK, was closely linked to the level of RNA-dependent ATPase activity observed in vitro for each protein. Moreover, the termination efficiency of four of the altered rho proteins at IS2 and lambda tR1 in vivo corresponded directly to the effect of these mutations on rho function at the E. coli trp t' terminator in vitro. We conclude that determinations of rho function in vitro accurately reflect its behavior in intracellular termination events.
Dombroski
A J
AJ
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Platt
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics; D001665:Binding Sites; D002874:Chromosome Mapping; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005686:Galactokinase / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D009154:Mutation; D010957:Plasmids; D012234:Rho Factor / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D012234:Rho Factor; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D005686:Galactokinase