3D Orbital Tracking of a DNA Locus during the Process of Transcription

Abstract

The temporal resolution currently achieved to track actively transcribing genes by means of fluorescence is of the order of 1-10s. We report on the application of 3D orbital tracking to a previously reported model system allowing to simultaneously label by means of fluorescence the genetic locus and the synthesized mRNA using the EGFP-labeled MS2 coat protein [1]. Our method, previously applied to the tracking of gene arrays in cultured cells [2], has a temporal resolution of 10-100ms, and additionally records the 3D position of the genetic locus by moving along a circular orbit the focused laser beam. Distinct regions of active transcription display a well defined spatial organization, corralling the denser part of the genetic locus. In most cases each region maintains a defined angle in the reference system of the orbit, and the transcriptional activities of different regions are not cross-correlated.The fluorescence time traces of each of these regions highlight the existence of slow (10-100s) transitions between distinct intensity values, corresponding to the timescale of a single mRNA dwell on the gene or to that of a transcription burst. We observe autocorrelation of the fluorescence intensity on timescales smaller than 1s. We relate these fast fluctuations to the faster kinetics of mRNA transcription, down to individual MS2-EGFP molecules binding to the newly transcribed mRNAs.Measurements of the size and shape of the genetic array by calculating the modulation of the first and second harmonic of the fluorescence along each orbit suggest that the gene's decondensation is not a necessary condition for transcription to occur.Work supported in part by NIH grants P50 GM076516 and P41 GM103540.[1] S. M. Janicki et al, Cell, 116, 683-698(2004).[2] V. Levi et al, Biophys. J., 89, 4275-4285(2005)