Structure-function relationships in Escherichia coli transcription termination protein Rho revealed by radiation target analysis.

High-energy electrons were used to measure the target sizes for inactivation of the RNA-dependent ATPase activity of Escherichia coli transcription termination factor Rho, for its ATP binding ability, and for its physical destruction. SDS-PAGE analysis of irradiated samples indicated that the target size for polypeptide destruction in the homohexameric enzyme is the dimer, indicating that energy transfer must occur from a hit subunit to one other subunit, although the subunits are not known to be linked by any covalent bonds. The ATP binding ability of Rho also inactivates as a dimer, a result that is consistent with the physical destruction target size. However, a single subunit as the ATP binding entity is not excluded. The RNA-dependent ATPase activity of Rho inactivates with the apparent target size of trimer to tetramer, indicating that interactions among the subunits of Rho are required for ATP hydrolysis. Rho hexamers are known to exchange subunits, although the identity of the exchanging unit is not known. Models in which this property of Rho is taken into account indicate that the closest fit to the experimental data is for an ATPase target size of a hexamer with dimers as the exchanging units, consistent with earlier chemical inactivation studies.

Mutations in an RNP1 consensus sequence of Rho protein reduce RNA binding affinity but facilitate helicase turnover.

Escherichia coli rho protein facilitates transcription termination by a mechanism that involves rho binding to the nascent RNA, activation of rho's RNA-dependent ATPase activity, and release of the mRNA from the DNA template. The initial step, formation of a rho-RNA complex, is mediated primarily by an RNA binding domain included within the amino-terminal 151 amino acids of rho protein. We have now identified one specific portion of this region that is involved in RNA binding, by photocross-linking and by site-directed mutagenesis. UV irradiation of rho-RNA complexes results in covalent attachment of the RNA to a single peptide in rho that apparently spans amino acids 45-100. Within this peptide is a ribonucleoprotein (RNP1) consensus sequence, Gly-Phe-Gly-Phe, that is present in many RNA-binding proteins. Mutagenesis of the phenylalanine residues in this consensus to leucine or alanine results in mutant proteins that are defective for RNA binding and have altered ATPase and RNA-DNA helicase activities. The weakened affinity but increased salt sensitivity of RNA binding by the mutant proteins suggests that they have lost more than just a set of nonionic interactions and are consistent with a change in the conformation of the RNA binding site. Whatever the changes, they appear localized primarily to the RNA binding domain because the mutants retain much of their RNA-dependent ATPase activity. We infer that the Phe residues themselves do not play a substantial role in the activation of ATP hydrolysis. Our results indicate that several different components of RNA interaction are required for rho activity and support a role for the RNP1 consensus region of rho in at least one specific aspect of RNA binding.