Gene expression in eukaryotic cells involves many steps and numerous components () [39••
]. Biochemical studies have identified most players and detailed the enzymatic nature of the process. Various hues of FPs allow multicolor labeling of DNA, RNA, and protein factors involved in gene expression. In addition, novel spectral properties such as photoswitching or fluorescent timers open the way for pulse-chase experiments at a single cell level. Currently it is possible to image three red colors (simultaneous imaging mOrange2 and TagRFP657, and asynchronous imaging of LSS-mKate2). Combination of RFPs with conventional blue/green and large Stokes shift GFPs could image as many as six colors in a single cell.
In , we have shown schematically the process of gene expression and how each step can be visualized. First, a specific gene locus on a chromosome can be tagged with DNA binding protein fused to FP by inserting recombinant DNA sequences carrying specific binding sites (such as Lac operator/repressor). Additionally, multiple mRNAs can be visualized in a single cell by incorporating a specific sequence recognized by an RNA binding protein labeled by FPs [41
]. When the gene is transcribed, multiple nascent transcripts accumulate and illuminate the transcription site.
Figure 3 The gene expression in eukaryotic cells involves many steps and numerous components. First transcription requires close cooperation between transcription factor, corregulator, mediator, chromatin remodeler, histone covalent modifier, and basal transcription (more ...)
In order to investigate mechanistic details, various factors that participate or regulate transcription can be labeled. Nuclear receptors (NR) are transcription factors that regulate gene expression in a ligand-dependent manner. Binding of agonist ligand triggers conformation changes of NR that leads to the recruitment of coactivators. Dual-color FFS has been successfully applied to study the concentration, mobility, and interactions of NR and its interaction with coactivators [36
]. The transcription dynamics are measured by applying FRAP or photoactivation to the transcription site. In this way, the residence time of various factors and dynamics of RNA polymerase has been measured [43•
]. It reveals surprisingly dynamic behavior and short binding times for most factors at the transcription site except the polymerase, which elongates the transcript. Novel photoswitching FPs will allow us to follow transcription initiation, elongation, and termination at the same time. MS2 labeled mRNA was tracked in the nucleus and showed that Brownian diffusion dictates the transport [45
]. By labeling the nuclear pore complex and applying super-registration microscopy, we and others have observed mRNA going through a single nuclear pore [26
]. Finally, the mRNA reaches cytoplasm and is translated. Fluorescent protein is commonly used as reporter for gene activity. For example, gene product tagged with fluorescent timers enables monitoring gene expression by conventional microscopy or flow cytometry [4