Fitting single-molecule data with the full stochastic model.

Experimental data show the fluctuation dynamics of gene constructs encoding an N-terminal 10X FLAG ‘Spaghetti Monster’ SM-tag (green) followed by a protein of interest and finally a 24X MS2 tag (red) in the 3’ UTR region. Three proteins were studied: A) H2B (orange), B) β-actin (blue) and C) KDM5B (violet). Middle figures show the simulated (colors) and measured (black) probability distributions for an mRNA to have a fluorescence intensity corresponding to i units of mature proteins (ump). Right images show the normalized auto-covariance function (G) calculated from experimentally measured (black error bars) and computationally simulated (colors) autocorrelation functions. Error bars in the experimental data and shadow bars in the simulated auto-covariance plots represent the standard errors of the mean. Elongation and initiation rates were obtained by parameter optimization, using the Hooke and Jeeves Algorithm ([29]). Optimized parameters and their uncertainties (see Methods) are provided in Eq 29.