Physical properties of the Escherichia coli transcription termination factor rho. 2. Quaternary structure of the rho hexamer.
Under approximately physiological conditions, the transcription termination factor rho from Escherichia coli is a hexamer of planar hexagonal geometry [Geiselmann, J., Yager, T. D., Gill, S. C., Calmettes, P., & von Hippel, P. H. (1992) Biochemistry (preceding paper in this issue)]. Here we describe studies that further define the quaternary structure of this hexamer. We use a combination of chemical cross-linking and treatment with mild denaturants to show that the fundamental unit within the rho hexamer is a dimer stabilized by an isologous (or pseudoisologous) bonding interface. Three identical dimers of rho interact via a second type of isologous bonding interface to yield a hexamer with C3 or D3 symmetry. Cross-linking and denaturation experiments definitely rule out C6 and C2 symmetry for the rho hexamer. Data from fluorescence quenching, lifetime, and energy transfer experiments also argue against C2 symmetry. The simplest symmetry assignment that is not contradicted by any experimental data is D3; thus we conclude that the rho hexamer has D3 symmetry. We also consider the positioning of the binding sites for RNA and ATP relative to the coordinate reference frame of the D3 hexamer. Fluorescence energy transfer data are presented and integrated with data from the literature to arrive at a self-consistent model for the quaternary structure of the rho hexamer.
Institute of Molecular Biology, University of Oregon, Eugene 97403.
J
J
Geiselmann
S E
SE
Seifried
T D
TD
Yager
C
C
Liang
P H
PH
von Hippel
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer; D004926:Escherichia coli / Q000737:chemistry*; D005454:Fluorescence Polarization; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011489:Protein Denaturation; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012234:Rho Factor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D012234:Rho Factor; D012313:RNA; D000255:Adenosine Triphosphate