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Resonant energy transfer from an optically excited donor molecule to a non-excited acceptor molecule residing nearby is widely used to detect molecular interactions in living cells. To date, resonant energy transfer has been used to obtain stoichiometric information, such as the number of proteins forming a complex, for a handful of proteins, but only after performing sequential scans of the emission wavelengths, excitation wavelengths, or sometimes both. During this lengthy process of measurement, the molecular makeup of a cellular region may change, limiting the applicability of resonant energy transfer to the determination of cellular averages. Here, we demonstrate a method for the determination of protein complex size, configuration, and spatial distribution in single living cells. It relies on a spectrally resolved two-photon microscope, a simple but competent theory, and a judicious selection of fluorescent tags. This approach eventually may lead to tracking the dynamics of individual molecular complexes inside living cells. The combination of spectrally resolved two-photon microscopy, fluorescent tags and appropriate theory makes it possible to determine the complex size, configuration and spatial distribution of proteins in single living cells. The findings made could lead to ways of tracking the cellular dynamics of individual molecular complexes.

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation. We show here that the essential FCP1 gene of S. cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo. Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo. This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro. The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S. cerevisiae generally requires CTD phosphatase.

Mutations that increase the low-level transcription of the Saccharomyces cerevisiae HIS4 gene, which results from deletion of the genes encoding transcription factors BAS1, BAS2, and GCN4, were isolated previously in SIT1 (also known as RPO21 , RPB1 , and SUA8 ), the gene encoding the largest subunit of RNA polymerase II (RNAPII). Here we show that sit1 substitutions cluster in two conserved regions of the enzyme which form part of the active site. Six sit1 mutations, affect region F, a region that is involved in transcriptional elongation and in resistance to α-aminatin. Four sit1 substitutions lie in another region involved in transcriptional elongation, region D, which binds Mg 2+ ions essential for RNA catalysis. One region D substitution is lethal unless suppressed by a substitution in region G and interacts genetically with PPR2 , the gene encoding transcription elongation factor IIS. Some sit1 substitutions affect the selection of transcriptional start sites at the CYC1 promoter in a manner reminiscent of that of sua8 ( sua stands for suppression of upstream ATG) mutations. Together with previous findings which indicate that regions D and G are in close proximity to the 3′ end of the nascent transcript and that region F is involved in the translocation process, our results suggest that transcriptional activation by the sit1 mutations results from alteration of the RNAPII active center.

We have determined the location of cis-acting elements that are important for the expression of RPO21 and RPO22, genes that encode the two largest subunits of RNA polymerase II (RNAPII) in Saccharomyces cerevisiae. A series of 5'-end deletions and nucleotide substitutions in the upstream regions of RPO21 and RPO22 were tested for their effect on the expression of lacZ fusions of these genes. Deletion of sequences from -723 to -693 in RPO21, which disrupted two Reb1p-binding sites and an Abf1p-binding site, resulted in a 10-fold decrease in expression. A T-rich region downstream of these sites was also important for expression. Deletion of sequences from -437 to -392 in the RPO22-upstream, which resulted in a 30-fold decrease in expression, indicated that the Reb1p- and Abf1p-binding sites in this region were important for RPO22 expression, as was a T-rich sequence immediately downstream of these sites. The RPO21 and RPO22 upstream regions were capable of interacting in vitro (gel-mobility-shift assays) with Reb1p and Abf1p. The similarities in the type and organization of elements in the upstream regions of RPO21 and RPO22 suggest that expression of these genes may be regulated coordinately.

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M(2) muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M(2) receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M(2) receptor to enhanced green fluorescent protein-M(2) receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n = 2, 0.495 +/- 0.019; n = 4, 0.202 +/- 0.010; n = 6, 0.128 +/- 0.006; n = 8, 0.093 +/- 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20-0.24, identifying the M(2) receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.

We have investigated the origin of the dielectric response of the plasma membrane of living yeast cells ( Saccharomyces cerevisiae ) by using radiofrequency dielectric spectroscopy. The cells were genetically engineered to overexpress in the membrane of yeast cells a G protein-coupled receptor – the Sterile2-α factor receptor protein (Ste2p) – fused to the green fluorescent protein (GFP). Presence of the Ste2-GFP proteins in the plasma membrane was confirmed by exciting the cells at 476 nm and observing with a confocal microscope the emission characteristic of the GFP from individual cells. The dielectric behavior of cells suspended in KCl solution was analyzed over the frequency range 40 Hz–110 MHz and compared to the behavior of control cells that lacked the ability to express Ste2p. A two-shell electrical cell model was used to fit the data starting from known structural parameters and adjustable electrical phase parameters. The best-fit value for the relative permittivity of the plasma membrane showed no significant difference between cells expressing Ste2p (1.63 ± 0.11) and the control cells (1.75 ± 0.16). This result confirmed earlier predictions that the dielectric properties of the plasma membrane in the radiofrequency range mostly reflect the properties of the hydrophobic layer of the membrane, which is populated by the hydrocarbon tails of the phospholipids and hydrophobic segments of integral membrane proteins. We discuss ways by which dielectric spectroscopy can be improved to be used for tag-free detection of proteins on the membrane.

Fluorescence Resonance Energy Transfer (FRET) - a process of nonradiative energy transfer from an optically excited molecule (donor, D) to an unexcited nearby molecule (acceptor, A) - is a powerful tool in studies of protein-protein interactions in living cells. FRET can be quantified, e.g., by measuring an increase in the donor fluorescence after inactivating the acceptor through photobleaching. In spite of its sheer simplicity, this method also introduces donor bleaching, which often complicates the interpretation of data. Correction methods are complicated by the fact that D photobleaching depends on whether a D molecule is free or coupled to an A molecule. In this communication we show that, instead of being a nuisance, donor bleaching actually can be harnessed to provide invaluable information about a population of interacting proteins in vivo. We present data on proteins tagged with fluorescent molecules, which indicate that donor and acceptor bleaching kinetics reveal quantitative information on the stoichiometry of proteinprotein interaction. Under appropriately chosen conditions, we are able to model the bleaching kinetics of D and A (both interacting and noninteracting) and determine the stoichiometry of the protein interaction. This provides a method for imaging protein complexes in living cells, which opens a way for testing the law of mass action in vivo.

We describe a fluorescence resonance energy transfer (FRET)-based method for finding in living cells the fraction of a protein population (alpha(T)) forming complexes, and the average number (n) of those protein molecules in each complex. The method relies both on sensitized acceptor emission and on donor de-quenching (by photobleaching of the acceptor molecules), coupled with full spectral analysis of the differential fluorescence signature, in order to quantify the donor/acceptor energy transfer. The approach and sensitivity limits are well suited for in vivo microscopic investigations. This is demonstrated using a scanning laser confocal microscope to study complex formation of the sterile 2 alpha-factor receptor protein (Ste2p), labelled with green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP respectively), in budding yeast Saccharomyces cerevisiae. A theoretical model is presented that relates the efficiency of energy transfer in protein populations (the apparent FRET efficiency, E(app)) to the energy transferred in a single donor/acceptor pair (E, the true FRET efficiency). We determined E by using a new method that relies on E(app) measurements for two donor/acceptor pairs, Ste2p-CFP/Ste2p-YFP and Ste2p-GFP/Ste2p-YFP. From E(app) and E we determined alpha(T) approximately 1 and n approximately 2 for Ste2 proteins. Since the Ste2p complexes are formed in the absence of the ligand in our experiments, we conclude that the alpha-factor pheromone is not necessary for dimerization.

Elongin is a transcription elongation factor that was first identified in mammalian systems and is composed of the three subunits, elongin A, B, and C. Sequence homologues of elongin A and elongin C, but not elongin B, were identified in the yeast genome. Neither yeast elongin A nor C sequence homologues was required for cell viability. The two gene products could be purified from yeast as a complex. A recombinant form of the complex, which could only be produced in bacteria if the gene products were co-expressed, was purified over several chromatographic steps. The complex did not stimulate transcription elongation by yeast RNA polymerase II. Using limited proteolysis, the N-terminal 144 residues of yeast elongin A were shown to be sufficient for interaction with yeast elongin C. The purified complex of yeast elongin C/elongin A(1-143) was analyzed using circular dichroism and nuclear magnetic spectroscopy. These studies revealed that yeast elongin A is unfolded but undergoes a dramatic modification of its structure in the presence of elongin C, and that elongin C forms a stable dimer in the absence of elongin A.

RNA polymerase II lacking the Rpb9 subunit uses alternate transcription initiation sites in vitro and in vivo and is unable to respond to the transcription elongation factor TFIIS in vitro. Here, we show that RPB9 has a synthetic phenotype with the TFIIS gene. Disruption of RPB9 in yeast also resulted in sensitivity to 6-azauracil, which is a phenotype linked to defects in transcription elongation. Expression of the TFIIS gene on a high-copy plasmid partially suppressed the 6-azauracil sensitivity of Deltarpb9 cells. We set out to determine the relevant cellular role of yeast Rpb9 by assessing the ability of 20 different site-directed and deletion mutants of RPB9 to complement the initiation and elongation defects of Deltarpb9 cells in vivo. Rpb9 is composed of two zinc ribbons. The N-terminal zinc ribbon restored the wild-type pattern of initiation start sites, but was unable to complement the growth defects associated with defects in elongation. Most of the site-directed mutants complemented the elongation-specific growth phenotypes and reconstituted the normal pattern of transcription initiation sites. The anti-correlation between the growth defects of cells disrupted for RPB9 and the selection of transcription start sites suggests that this is not the primary cellular role for Rpb9. Genome-wide transcription profiling of Deltarpb9 cells revealed only a few changes, predominantly in genes related to metabolism.

In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy. In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII. Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium. We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS. Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steady-state amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme. We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery.

Only a minimum generally can be placed on the size of oligomers formed by G protein-coupled receptors. Attempts to obtain an explicit estimate have not been persuasive, owing in part to their reliance on questionable models. We therefore have developed a quantitative method based on fluorescence resonance energy transfer (FRET), which we have applied to oligomers of M2 receptors tagged at the N-terminus with enhanced green or yellow fluorescent protein (eGFP-M2 or eYFP-M2) and co-expressed in Chinese hamster ovary cells. The approach is based on the relationship between apparent FRET efficiency and the ‘pair-wise’ FRET efficiency (E) between a single donor and a single acceptor in an oligomer of specified size (n). Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were measured by donor-dequenching and acceptor-sensitized emission at different ratios of eYFP-M2 to eGFP-M2. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within the oligomer to obtain fitted values of E as follows: n=2, 0.473±0.011; n=4, 0.206±0.009; n=6, 0.132±0.005; n=8, 0.100±0.002. The pair-wise FRET efficiency determined independently by fluorescence lifetime imaging was 0.206. The M2 receptor therefore can be identified as a tetramer, in agreement with the results of a parallel study in which the oligomeric size has been estimated from FRET efficiencies measured at the level of single pixels.