Three-dimensional reconstruction of transcription termination factor rho: orientation of the N-terminal domain and visualization of an RNA-binding site.

The Escherichia coli rho transcription termination protein is a hexameric helicase, and is believed to function by separating an RNA-DNA hybrid. Unlike hexameric DNA helicases, where a single strand of DNA passes through the central channel, it has been proposed that the RNA wraps around the outside of the ring. We have generated a three-dimensional reconstruction of rho, and localized a tRNA molecule bound to the primary RNA-binding site to the outside of the ring. An atomic structure of the N-terminal domain of rho fits into our reconstruction uniquely, with the residues involved in RNA-binding on the outside of the ring. Although rho shares a common structural core with the F1-ATPase and other hexameric helicases, there has been a divergence in function due to rho's N-terminal domain, which has no homology to other helicases.

Structure and assembly of the Escherichia coli transcription termination factor rho and its interaction with RNA. I. Cryoelectron microscopic studies.

Cryoelectron microscopy has been used to visualize the Escherichia coli transcription termination protein rho in a vitreously frozen state, without the use of strains, fixatives or other chemical perturbants. In the absence of RNA cofactor, a variety of structures are observed, reflecting the heterogeneity of complexes formed by rho at protein concentrations near the physiological range (3 to 10 microM). One of the most common structural motifs we see is a six-membered ring of rho subunits (present as either a closed or "notched" circle), which corresponds to the predominant hexameric association state of the protein. Also visible are smaller oligomeric structures, present as curved lines of rho subunits, which probably represent the lower association states of the protein that coexist with the hexamer at these protein concentrations. Addition of oligomers of ribocytosine (rC) of defined lengths (23-mers and 100-mers) results in the generation of more homogeneous populations of rho oligomers. In the presence of (rC)23, all identifiable particles appear either as closed or as notched hexameric circles. A small fraction of these particles are of visibly higher density, and are identified with the dodecamers expected as a subpopulation of rho under these conditions. Binding of (rC)100, an oligomer of length greater than that needed to span the entire hexamer binding site, results in a uniform population of closed circular hexamers. In some images additional features are visible at either the centers or the peripheries of the particles. These features may correspond to the excess length of the rC strands bound to the hexamers. The distributions of particles observed under the various experimental conditions used correlate well to those deduced from physical biochemical studies Seifried et al., accompanying paper).