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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.

Transcription 3' movement of the protein along the nascent RNA to elongation complexes paused at specific Rho-dependent termination sites on the DNA template. Rho then engages its ATPase-dependent RNA-DNA helicase activity to release the RNA from the transcription complex (5). The sequence specificity of Rho-dependent termination sites appears to reflect the extended dwell-time of the elongation complex at these positions (14-16). This extensive pausing allows Rho proteins that are translocating along the nascent RNA to "catch up" with the transcription complex at these sites, with the resultant termination efficiency depending on the "kinetic coupling" ofthe rate ofmovement ofRho along the nascent transcript and of RNA polymerase along the DNA template (1,17,18).The development of this view of Rho function has been paralleled by progress in elucidating its structural and enzymatic properties. It has been shown that Rho exists under physiological conditions as a hexamer of identical subunits (19-23), organized as a trimer of asymmetric dimers with overall D3 symmetry (24). This symmetry is reflected functionally in the presence of three strong and three weak ATP (substrate) and three strong and three weak RNA (cofactor) binding sites per Rho hexamer (25)(26)(27)(28) (37,38), and subunit interaction sites have been proposed in the C-terminal region (35). Rho monomers associate to form a hexamer with D3 symmetry under physiological conditions.

Interactions between proteins and nucleic acids are important in the fundamental cellular processes that drive replication, recombination, dynamic alteration and repair of DNA, transcription and processing of RNA, synthesis of proteins, and regulation of enzyme activities. As part of an effort to develop a general, sensitive mass spectrometric strategy for the characterization of protein-nucleic acid interactions, we have used matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry to analyze protein-nucleic acid complexes that have been covalently crosslinked by ultraviolet (UV) light. In general, the application of MALDI mass spectrometric techniques to studies of UV-induced crosslinking of nucleoprotein complexes is demonstrated to be feasible. Specifically, MALDI mass analysis was used to determine the molecular weights of the phage T4 gene 32 protein (gp32) crosslinked to the oligonucleotide (dT)20, and the Escherichia coli transcription termination factor rho, photoaffinity labeled with 4-thio-uridine-diphosphate (4sUDP). The covalent gp32:(dT)20 complex is readily detected at a concentration of 1-2 microM in 1 microL of an unpurified solution of reactants that has been exposed to a single, 266 nm UV laser pulse. Mass spectrometric molecular weight determinations of the covalent rho:4sUDP complex add directness and specificity to the ATPase inactivation assay normally used to monitor the formation of 4sUDP photoaffinity labeled rho. It is found that successful MALDI mass spectrometry of protein-nucleic acid complexes is as critically dependent on the choice of solvents and additives as it is on the primary matrix compound.