Abstract
Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.
Highlights
The genome is a complex and very dense viscoelastic polymer matrix, and it is difficult to study individual components of the gene expression machinery with conventional light microscopy
Since the publication of one of the seminal reviews on super-resolution microscopy (SRM) in 2010 [11], twelve years have passed during which the expectation of authors that “it will still take time and further engineering until technical developments find their way into commercial systems” has become a reality
As we have reviewed here the examples of some of the single-molecule localization microscopy (SMLM) applications to monitor the dynamics of transcription factors (TFs) and RNA polymerase II (RNAPII) in the living cell, in the past decade we have witnessed rapid progress in the 4D single-molecule kinetic studies in living cells
Summary
The genome is a complex and very dense viscoelastic polymer matrix, and it is difficult to study individual components of the gene expression machinery with conventional light microscopy. The optical resolution of the conventional light microscopy is limited by the diffraction of light and allows to distinguish the objects only if they are ~200 nm apart. This ~200 nm resolution limit of the classical light microscope is given by the nature of light and results from the fundamental laws of physics. Reducing the wavelength of the light used for imaging and/or increasing the NA of the objective lens improves the spatial resolution of a conventional light microscope. Rayleigh elaborated on the diffraction limit, and in 1896, postulated that the smallest resolvable distance (dmin ) between two points is proportional to the wavelength in the vacuum of the light used for imaging and inversely proportional to the NA with a factor of. We will first focus on SMLM applications to studying the dynamics of transcription factors, and on the RNAPII itself
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