Dynamics of transcription and mRNA export

Abstract

Understanding the different molecular mechanisms responsible for gene expression has been a central interest of molecular biologists for several decades. Transcription, the initial step of gene expression, consists of converting the genetic code into a dynamic messenger RNA that will specify a required cellular function following translocation to the cytoplasm and translation. We now possess an in-depth understanding of the mechanism and regulations of transcription. By contrast, an understanding of the dynamics of an individual gene's expression in real time is just beginning to emerge following recent technological developments.

Figure 1

Tandem gene arrays for the analysis of nuclear factor dynamics in living cells. The establishment of integrated tandem gene arrays in the genome provides a platform on which a signal from a fluorescently labeled DNA- or RNA-binding protein can be amplified, detected and analyzed kinetically. (a) The locus of integration occurs randomly in the genome and (b) many copies of the gene are inserted in tandem at this locus. (c) Different types of gene arrays have been used. (1) lac operator repeats (lacO) are bound by the lac repressor protein (LacI), which can either be tagged with a fluorescent protein to mark the locus of integration or can be fused to a protein of interest, thus tethering it to the chromatin. (2) Tandem arrays of promoters can serve for the analysis of transcription factor (TF) binding dynamics. (3) The production of mRNA can be followed using gene arrays that transcribe mRNAs containing MS2 repeats, which are bound by the YFP–MS2 protein. The kinetics of RNA pol II or RNA processing factors can also be analyzed on such arrays. (d) Photobleaching methods such as FRAP are used to measure transcription-factor binding to gene arrays. (e) The onset of transcription can be measured upon cellular stimulation (red line shows a classical activation with saturation; orange line shows activation with a negative feedback).

Figure 2

Visualizing the binding of nuclear factors to tandem gene arrays. Human cells containing a tandem gene array (200 copies) were used for real-time detection of the recruitment of different factors to the site of active transcription. The tetracycline-inducible gene module gives rise to an intron-containing mRNA coding for a peroxisome-targeted CFP (cyan fluorescent protein) and containing 24 MS2 repeats in the 3′UTR. Each gene module is flanked by 256 repeats of the lacO binding site. (a,b,c) After induction of transcription, the gene locus is detected by the binding of the CFP–LacI protein to the lacO repeats and the peroxisome-targeted CFP protein product is observed in the cytoplasm. (d) The production of the mRNA at the gene locus is visualized by the binding of the yellow fluorescent protein (YFP)–MS2 protein to the MS2 stem-loops in the mRNA. (e) The recruitment of the transcription machinery to this active site of transcription is seen by the enrichment of YFP–RNA pol II at the locus. (f) The pre-mRNA splicing factor YFP–SF2/ASF is also enriched at the locus. (g,h,i) Merge. (Adapted from Janicki et al. [ 44^••]).