Single molecule studies of RNA polymerase II transcription in vitro

Horn, Abigail E.; Goodrich, James A.; Kugel, Jennifer F.

doi:10.4161/trns.27608

Cited by 4 publications

(1 citation statement)

References 35 publications

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“…Moreover, single-molecule techniques are increasingly being used to study genome maintenance processes in eukaryotes, expanding our understanding of the intricate molecular mechanisms governing fundamental biological processes like DNA replication ,− and transcription. , This has allowed for the revelation of new mechanisms underlying genome dynamics, cellular function, and disease pathology, with potential applications, from foundational research to the design of innovative therapeutic strategies in drug development.…”

Section: Conclusion and Perspectivementioning

confidence: 99%

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

Sharma,

van Oijen,

Spenkelink

et al. 2024

Chemical & Biomedical Imaging

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Genome maintenance comprises a group of complex and interrelated processes crucial for preserving and safeguarding genetic information within all organisms. Key aspects of genome maintenance involve DNA replication, transcription, recombination, and repair. Improper regulation of these processes could cause genetic changes, potentially leading to antibiotic resistance in bacterial populations. Due to the complexity of these processes, ensemble averaging studies may not provide the level of detail required to capture the full spectrum of molecular behaviors and dynamics of each individual biomolecule. Therefore, researchers have increasingly turned to single-molecule approaches, as these techniques allow for the direct observation and manipulation of individual biomolecules, and offer a level of detail that is unattainable with traditional ensemble methods. In this review, we provide an overview of recent in vitro and in vivo single-molecule imaging approaches employed to study the complex processes involved in prokaryotic genome maintenance. We will first highlight the principles of imaging techniques such as total internal reflection fluorescence microscopy and atomic force microscopy, primarily used for in vitro studies, and highly inclined and laminated optical sheet and super-resolution microscopy, mainly employed in in vivo studies. We then demonstrate how applying these single-molecule techniques has enabled the direct visualization of biological processes such as replication, transcription, DNA repair, and recombination in real time. Finally, we will showcase the results obtained from super-resolution microscopy approaches, which have provided unprecedented insights into the spatial organization of different biomolecules within bacterial organisms.

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Section: Conclusion and Perspectivementioning

confidence: 99%

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

Sharma,

van Oijen,

Spenkelink

et al. 2024

Chemical & Biomedical Imaging

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Observing Single RNA Polymerase Molecules Down to Base-Pair Resolution

Chakraborty¹,

Meng²,

Block³

2016

Optical Tweezers

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During transcriptional elongation, RNA polymerases (RNAP) employ a stepping mechanism to translocate along the DNA template while synthesizing RNA. Optical trapping assays permit the progress of single molecules of RNA polymerase to be monitored in real time, at resolutions down to the level of individual base pairs. Additionally, optical trapping assays permit the application of exquisitely controlled, external forces on RNAP. Responses to such forces can reveal details of the load-dependent kinetics of transcriptional elongation and pausing. Traditionally, the bacterial form of RNAP from E. coli has served as a model for the study of transcriptional elongation using optical traps. However, it is now feasible to perform optical trapping experiments using the eukaryotic polymerase, RNAPII, as well. In this report, we describe the methods to perform optical trapping transcriptional elongation assays with both prokaryotic RNAP and eukaryotic RNAPII. We provide detailed instructions on how to reconstitute transcription elongation complexes, derivatize beads used in the assays, and perform optical trapping measurements.

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Monitoring transcriptional activity by RNA polymerase II in vitro using single molecule co-localization

Goodrich

Kugel

2019

Methods

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Single molecule studies of RNA polymerase II transcription in vitro

Cited by 4 publications

References 35 publications

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

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

Observing Single RNA Polymerase Molecules Down to Base-Pair Resolution

Monitoring transcriptional activity by RNA polymerase II in vitro using single molecule co-localization

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