The Nestled Jewel Of I-BET-762
The remaining jumps were interpreted as RNA production times, from which intervals between transcription events were calculated. Finally, censored intervals were calculated as the time from the last RNA production in a cell until the last time at which a jump could have been observed (i.e. until 5 min prior to cell division or the end of the timeseries). This removes the possibility of false selleck products positives while not affecting the distribution of intervals. This method, when first proposed, made two assumptions on the fluorescence of MS2-GFP tagged RNAs (named ��spots��). Importantly, both assumptions were recently shown to be valid.42 First, an individual spot is bound sufficiently rapidly by MS2-GFPs such that its fluorescence intensity, when first detected, is already within the range of fluorescence of fully formed MS2-GFP-RNA spots (when taking one image per minute). In other words, the spot intensity of a newly transcribed RNA jumps from 0 to ��full�� in diglyceride site region of the target RNA is ?60 s. Provided that MS2-GFP binding to its RNA-binding sites is fast, there will therefore be a maximum of one timepoint at which the fully transcribed target RNA may have reduced fluorescence. Since MS2-GFP is produced in excess in the cell and its binding affinity is strong (dissociation constant of ?0.04 nM43), most binding sites will be saturated very shortly after being produced. I-BET-762 ic50 In agreement with ref. 42, no gradual increase in spot fluorescence was observed around the time of the first appearance of a spot. Second, once formed, MS2-GFP-RNA spots, as well as their fluorescence, are resistant to degradation for the duration of our measurements (2 h). This was shown by measurements of the dissociation rate of MS2 coat proteins from their RNA binding sites (on the order of several hours43), and by measurements of the lifetimes of the fluorescence of MS2-GFP tagged RNAs kept under observation for more than 2 h.1,2,5,42,44 Relevantly, no detectable decrease in fluorescence was observed during this time.42 2.7. Model of transcription initiation We first consider a model that allows for RNA production dynamics to range from sub-Poissonian to super-Poissonian, given the results from genome-wide studies of the variability in RNA numbers27,45 and from studies of the transcription dynamics of individual genes.2,4,5,17,20 The features of the model that allow it to reproduce these numbers are based on processes known to occur during transcription initiation in E. coli (e.g. the open complex formation16,22,23 and an ON/OFF mechanism16,19). Then, based on our novel empirical data and methodology, we aim to obtain the most parsimonious version of the model that fits the data for a given promoter.
