New mechanisms that regulate Saccharomyces cerevisiae short peptide transporter achieve balanced intracellular amino acid concentrations.

The budding yeast Saccharomyces cerevisiae is able to take up large quantities of amino acids in the form of di- and tripeptides via a short peptide transporter, Ptr2p. It is known that PTR2 can be induced by certain peptides and amino acids, and the mechanisms governing this upregulation are understood at the molecular level. We describe two new opposing mechanisms of regulation that emphasize potential toxicity of amino acids: the first is upregulation of PTR2 in a population of cells, caused by amino acid secretion that accompanies peptide uptake; the second is loss of Ptr2p activity, due to transporter internalization following peptide uptake. Our findings emphasize the importance of proper amino acid balance in the cell and extend understanding of peptide import regulation in yeast.

Effect of loop composition on the stability and folding kinetics of RNA hairpins with large loops.

RNA hairpins are ubiquitous structural elements in biological RNAs, where they have the potential to regulate RNA folding and interactions with other molecules. There are established methods for predicting the thermodynamic stability of an RNA hairpin, but there are still relatively few detailed examinations of the kinetics of folding. Nonetheless, several recent studies indicate that hairpin folding does not proceed via a simple two-state model. Here, we monitor fluorescence from hairpins constructed as molecular beacons in ensemble, fluorescence correlation spectroscopy, and stopped-flow experiments to describe the folding of RNA hairpins with long (15 nucleotide) loops. Our results show that folding of these hairpins occurs through more than two states and that the mechanism of folding includes a fast intermediate phase observed on the tens of microseconds time scale and a slow phase, attributed to formation of the native folded hairpin loop and stem, observed on the milliseconds time scale. The composition of the RNA loop determines the time scale of intermediate and native folded states. Hairpins with a polyuracil loop sequence exhibit slower relaxation of the intermediate state and faster relaxation of the native folded state when compared to that of hairpins with cytosine or adenine in the loop. We hypothesize this composition dependence could be attributed to nucleobase stacking in cytosine and adenine containing regions of the loop, which would be absent in hairpins containing polyuracil loops. Such base stacking could destabilize the intermediate folds, thereby speeding the relaxation of the intermediate relative to similar sized hairpins with no base stacking in the loop. Likewise, the lower intermediate stability could prolong the relaxation of the native folded state.

Confidence intervals for concentration and brightness from fluorescence fluctuation measurements.

The theory of photon count histogram (PCH) analysis describes the distribution of fluorescence fluctuation amplitudes due to populations of fluorophores diffusing through a focused laser beam and provides a rigorous framework through which the brightnesses and concentrations of the fluorophores can be determined. In practice, however, the brightnesses and concentrations of only a few components can be identified. Brightnesses and concentrations are determined by a nonlinear least-squares fit of a theoretical model to the experimental PCH derived from a record of fluorescence intensity fluctuations. The χ(2) hypersurface in the neighborhood of the optimum parameter set can have varying degrees of curvature, due to the intrinsic curvature of the model, the specific parameter values of the system under study, and the relative noise in the data. Because of this varying curvature, parameters estimated from the least-squares analysis have varying degrees of uncertainty associated with them. There are several methods for assigning confidence intervals to the parameters, but these methods have different efficacies for PCH data. Here, we evaluate several approaches to confidence interval estimation for PCH data, including asymptotic standard error, likelihood joint-confidence region, likelihood confidence intervals, skew-corrected and accelerated bootstrap (BCa), and Monte Carlo residual resampling methods. We study these with a model two-dimensional membrane system for simplicity, but the principles are applicable as well to fluorophores diffusing in three-dimensional solution. Using simulated fluorescence fluctuation data, we find the BCa method to be particularly well-suited for estimating confidence intervals in PCH analysis, and several other methods to be less so. Using the BCa method and additional simulated fluctuation data, we find that confidence intervals can be reduced dramatically for a specific non-Gaussian beam profile.

Revival of high-order fluorescence correlation analysis: generalized theory and biochemical applications.

Analysis of high-order correlations in fluorescence fluctuation spectroscopy was developed in the late 1980s but since then has been replaced by alternative brightness analysis methods. However, high-order correlation has important advantages in many experiments. We present a new cumulant-based formalism of high-order correlation that greatly simplifies data analysis. The new formalism is used to derive general expressions for variance of high-order correlations that show good agreement with experiment in a model system of fluorescently labeled DNA oligomers. A simulation of binary systems in which both diffusion time and brightness are varied illustrates clearly that high-order analysis has better sensitivity to brightness than fluorescence correlation spectroscopy (FCS). These results have implications for analysis of isomerization reactions and dual-beam FCS with flow. We also demonstrate that high-order correlations can detect photobleaching in the observation volume. The application of this formalism to many FCS-based experiments allows more accurate analysis in addition to describing more molecular parameters.