Structure and assembly of the Escherichia coli transcription termination factor rho and its interactions with RNA. II. Physical chemical studies.
Transcription termination factor rho from Escherichia coli is comprised of a hexamer of identical protein monomers. Hydrodynamic and light-scattering studies have shown the fully assembled rho to be a doughnut-shaped structure. Semi-denaturing gels, protein crosslinking, and spectroscopic studies, as well as other functional and binding determinations have established that the rho hexamer displays D3 symmetry (i.e. it exists as a trimer of dimers). In the accompanying paper we visualize rho directly in the absence of cofactor and show that binding of RNA it into the hexameric form. In this paper we examine the pathway and association constants involved in rho oligomer assembly. Sedimentation and fluorescence-detected size exclusion chromatography are used to demonstrate three steps in the assembly process. These steps can be differentiated by subunit association affinity and kinetic properties. The kinetics of the monomer-dimer equilibrium are fast and an apparent association constant of 1.3 x 10(6) M-1 is measured for this process. In contrast, the dimer-tetramer and tetramer-hexamer association processes appear to be slower (of the order of seconds) and to involve association constants that are smaller than that of the monomer-dimer reaction. This behaviour is consistent with a hexamer of D3 symmetry. Such a particle displays two kinds of subunit interactions; one associated with an intra-dimer A:A interface and the other with an inter-dimer B:B interface. The closure of the circular hexamer does not appear to contribute additional free energy to the assembly process. Fluorescence and sedimentation studies show the association steps to be sensitive to salt concentration. Consistent with earlier work, we find that assembly to the hexameric state is driven by RNA binding.
Seifried
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SE
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Bjornson
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KP
von Hippel
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PH
D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D046911:Macromolecular Substances; D011066:Poly C / Q000378:metabolism*; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000378:metabolism*; D012234:Rho Factor / Q000737:chemistry / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism
D046911:Macromolecular Substances; D012329:RNA, Bacterial; D012234:Rho Factor; D011066:Poly C; D012965:Sodium Chloride