Probing steps in DNA transcription using single-molecule methods

Abstract

Transcriptional regulation is one of the key steps in determining gene expression. Diverse single-molecule techniques have been applied to characterize the stepwise progression of transcription, yielding complementary results. These techniques include, but are not limited to, fluorescence-based microscopy with single or multiple colors, force measuring and manipulating microscopy using magnetic field or light, and atomic force microscopy. Here, we summarize and evaluate these current methodologies in studying and resolving individual steps in the transcription reaction, which encompasses RNA polymerase binding, initiation, elongation, mRNA production, and termination. We also describe the advantages and disadvantages of each method for studying transcription.

Insight into Single-Molecule Imaging Techniques for the Study of Prokaryotic Genome Maintenance

“…Altogether, these studies have highlighted the complexity involved in tracking the movement of individual RNAP molecules along the DNA during transcription initiation and elongation. These findings challenge the traditional view of transcription as a smooth and continuous process . Instead, they show that transcription is a highly dynamic process, which undergoes 3D-diffusion during the initial stages while searching for promoter sites, followed by 1D-sliding during the transcription elongation phase along the DNA template.…”

“…These findings challenge the traditional view of transcription as a smooth and continuous process. 137 Instead, they show that transcription is a highly dynamic process, which undergoes 3D-diffusion during the initial stages while searching for promoter sites, followed by 1Dsliding during the transcription elongation phase along the DNA template. These revelations underscore the multifaceted nature of transcriptional regulation and provide a solid foundation for further exploration into the molecular mechanisms governing gene expression.…”

EPR Spectroscopy Provides New Insights into Complex Biological Reaction Mechanisms

“…In general, FRET measurements are sufficiently accurate to describe kinetic parameters, overall mechanistic transitions, 7 and time scales, yet they cannot explain the fundamental mechanical driving forces nor provide accurate topological changes of structural rearrangements, data which are essential for following complex biological reaction mechanisms. 7 Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) are becoming major tools for determining protein structures at high-resolution. 8 Currently, the main advantage of cryo-EM/ET is that structures of large complexes or membrane proteins collected by these approaches can be resolved.…”

“…The power of FRET lies in its ability to report intermolecular interactions at the nanometer scale (1–10 nm). In general, FRET measurements are sufficiently accurate to describe kinetic parameters, overall mechanistic transitions, and time scales, yet they cannot explain the fundamental mechanical driving forces nor provide accurate topological changes of structural rearrangements, data which are essential for following complex biological reaction mechanisms . Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) are becoming major tools for determining protein structures at high-resolution .…”

Head-on and co-directional RNA polymerase collisions orchestrate bidirectional transcription termination

“…Single-molecule techniques are powerful tools to study dynamic biomolecular processes and have greatly aided our understanding of the mechanism of transcription and its regulation [reviewed in (Bai et al, 2006; Dangkulwanich et al, 2014; Larson et al, 2011; Lee and Myong, 2021; Wang and Greene, 2011)]. The two major classes of single-molecule approaches provide complementary information: fluorescence-based detection reveals the composition of transcription complexes [e.g.…”