Figure 6. Quantifying mRNA synthesis in vivo.

(a–s) FRAP (a–i) and loss of fluorescence after photoactivation (k–s) at the transcription site of the MS2-labeled mRNA. a,k show differential interference contrast images of live cells. In b,l, RFP-LacI labels gene locus. In c,m, dotted circle indicates photobleached and photoactivated regions, respectively. d,n show bleached MS2-GFP and activated paGFP-MS2, respectively. e–i show MS2-GFP recovery and o–s show paGFP-MS2 release from transcription site monitored for 10 min. Scale bars, 5 µm. (j,t) Normalized locus recovery (j) or loss in fluorescence (t) (black dots; n = 10). Curves show best-fit solutions for mathematical model (see equation) with single exponential (red) or two exponentials (blue), and inset tables list the resulting parameters (also see residuals in lower chart) along with the Akaike information criterion (AIC)⁵⁹ and the Bayes-Schwarz information criterion (BIC)⁶⁰. See Figure 2 for details. Both data sets require two exponentials, as the residuals are not randomly distributed with one exponential. When the MS2 photoactivation data (t) are fit to a single-exponential function, all the residuals for t < 200 s are negative and all those for t > 200 s are positive. If the two fits (j and t) are constrained to use the same Eigen values, the resulting mean residence times are 238 s and 34 s (see also Table 1). Error bars show s.e.m.