Quantification of protein-protein interactions using fluorescence polarization.

Quantitative determinations of the dissociation constants of biomolecular interactions, in particular protein-protein interactions, are essential for a detailed understanding of the molecular basis of their specificities. Fluorescence spectroscopy is particularly well suited for such studies. This article highlights the theoretical and practical aspects of fluorescence polarization and its application to the study of protein-protein interactions. Consideration is given to the nature of the different types of fluorescence probes available and the probe characteristics appropriate for the system under investigation. Several examples from the literature are discussed that illustrate different practical aspects of the technique applied to diverse systems.

A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho.

Transcription termination protein Rho of Escherichia coli interacts with newly synthesized RNA chains and brings about their release from elongation complexes paused at specific Rho-dependent termination sites. Rho is thought to accomplish this by binding to a specific Rho "loading site" on the nascent RNA and then translocating preferentially along the transcript in a 5'-->3' direction. On reaching the elongation complex, Rho releases the nascent RNA by a 5'-->3' RNA.DNA helicase activity. These translocation and helicase activities are driven by the RNA-dependent ATPase activity of Rho. In this paper we propose a mechanism for these processes that is based on the structure and properties of the Rho protein. Rho is a hexamer of identical subunits that are arranged as a trimer of asymmetric dimers with D3 symmetry. The binding of ATP and RNA to Rho also reflects this pattern; the Rho hexamer carries three strong and three weak binding sites for each of these entities. The asymmetric dimers of Rho correspond to functional dimers that can undergo conformational transitions driven by ATP hydrolysis. We propose that the quaternary structure of Rho coordinates the ATP-driven RNA binding and release processes to produce a biased random walk of the Rho hexamer along the RNA, followed by RNA.DNA helicase activity and transcript release. The proposed model may have implications for other hexameric DNA.DNA, RNA.DNA, and RNA.RNA helicases that function in replication and transcription.

Direct observation of UV-crosslinked protein-nucleic acid complexes by matrix-assisted laser desorption ionization mass spectrometry.

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.

ATPase activity of transcription-termination factor rho: functional dimer model.

Transcription-termination factor rho of Escherichia coli functions as an RNA-dependent ATPase that causes transcript release at specific rho-dependent termination sites on the DNA template. Rho exists as a hexagon of identical subunits, physically organized as a trimer of dimers with D3 symmetry. The structural asymmetry of the dimer is reflected in the binding properties of rho; each dimer has a strong and a weak binding site for both the ATP substrate and the RNA cofactor. Here we use homopolynucleotides in competition and complementation experiments to characterize the ATPase activation properties of the cofactor binding sites of the functional rho dimer. We show that (i) no ATPase activity is observed unless both the high- and the low-affinity cofactor binding sites of the functional rho dimer are occupied; (ii) saturating levels of poly(rC), poly(rC) in combination with poly(rU), or poly(rU) alone can fully activate the ATPase of rho; and (iii) poly(dC) can serve as a fully competitive inhibitor of half of the ATPase activity of rho when one of the cofactor sites is filled with poly(rC). These observations lead to a set of phenomenological rules that describe the cofactor dependence of the ATPase activation of the functional dimer of rho and help to define a mechanistic basis for interpreting rho function in termination.

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.

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).

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.

Fluorescent modification of the cysteine 202 residue of Escherichia coli transcription termination factor rho.

The lone cysteine residue (Cys-202) of transcription termination factor rho has been modified with the sulfhydryl-specific dyes 5-iodoacetamidofluorescein and 5-(2-[iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid. Labeling with both dyes is specific for the Cys-202 residue and is at least 90% complete. Rho protein is an RNA-dependent ATPase and exists as a hexamer of identical subunits in its activated (RNA-liganded) form. We find that chemical modification of rho at Cys-202 does not significantly change the properties of the protein; subunit assembly, RNA binding, and poly(rC)-activated ATP hydrolysis are all relatively unperturbed by the covalent attachment of these fluorescent moieties. On the other hand, the spectral, quenching, and anisotropy properties of the fluorescent groups are all significantly modified by attachment to the protein. No energy transfer is seen between fluorescein-labeled subunits within rho hexamers, indicating that the Cys-202 residues on these subunits are located at least 40 A apart. These fluorescently labeled rho molecules should represent useful probes to study the conformations and inter- and intrasubunit geometries of this termination factor at various stages of its interaction with nascent RNA transcripts.