We present key examples of the use of single-molecule approaches to study transcription, translation, splicing and replication.
We highlight the particular advantages of using single-molecule approaches for the study of genome processing.
We provide an overview of the force manipulation and fluorescent techniques used to study genomic processes.
We highlight how single-molecule studies of transcription have provided novel insights into initiation, elongation and termination.
We discuss how in the field of translation, single-molecule methods have been used to dissect aspects of initiation, elongation, termination and protein folding.
Novel single-molecule fluorescence assays have allowed studies of splicing and nuclear export in unprecedented detail.
We review how single-molecule techniques provided surprising insights on the stoichiometry and dynamics of the replisome.
We conclude with an overview of challenges and future directions in the application of approaches to genomic processes.
To understand genomic processes such as transcription, translation or splicing, we need to be able to study their spatial and temporal organization at the molecular level. Single-molecule approaches provide this opportunity, allowing researchers to monitor molecular conformations, interactions or diffusion quantitatively and in real time in purified systems and in the context of the living cell. This Review introduces the types of application of single-molecule approaches that can enhance our understanding of genome function.
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This work was supported by the European Science Foundation through a European Young Investigators (EURYI) grant to N.H.D. and by the Netherlands Organisation for Scientific Research through grants to N.H.D. and J.L. We thank anonymous referees for useful feedback and D. Grünwald for a critical reading of the manuscript. J. Kerssemakers is thanked for the visual layout of Figure 5. We acknowledge the many research efforts by groups in the field of genome processing and regret that owing to space limitations it was not possible to cite a larger number of high-quality works.
The authors declare no competing financial interests.
A multi-protein complex that carries out DNA replication.
- Tethered particle motion
(TPM). A single-molecule technique that uses tethered beads to study biological molecules in the absence of any externally applied force. Changes in the average position of the bead can report on changes in tether length and hence on enzyme activity.
- Evanescent waves
Parallel optical waves with an exponentially decaying intensity that occur near a surface when incident light impinges at an angle greater than the critical angle for refraction.
- Rayleigh criterion
Quantifies the minimum resolvable distance between two objects that fluoresce at the same wavelength. This distance equals roughly half the wavelength of light.
- RNA polymerase holoenzyme
The initiation complex composed of the RNA polymerase core enzyme and the σ-initiation factor.
One of the initiation factors that can bind to Escherichia coli RNA polymerase during initiation to allow it to recognize a specific promoter sequence.
The most common and most widely studied initiation factor that can bind to Escherichia coli RNA polymerase during initiation to allow it to recognize a specific promoter sequence.
- Elongation factor G
(EF-G). A factor that provides the bacterial ribosome with the necessary energy (derived from GTP hydrolysis) required to translocate along the mRNA.
- Ribosomal A, P and E sites
The aminoacyl, peptidyl and exit sites of the ribosome, respectively, which are the three different binding sites of tRNAs.
Proteins that comprise the nuclear pore complex.
- Super-registration microscopy
The use of fluorescence microscopy to localize fluorescent probes as described in Box 2 in a way that accurately registers the relative positions of labels that fluoresce in different colours.
A description of how the chemical reactions driving biological processes, such as ATP hydrolysis in a molecular motor, are coupled to mechanical motion: for example, translocation along a nucleic acid template.
A protein complex consisting of a helicase and primase that is responsible for the synthesis of RNA primers during DNA replication.
- RNA aptamers
RNA molecules that specifically bind to a target molecule.
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Dulin, D., Lipfert, J., Moolman, M. et al. Studying genomic processes at the single-molecule level: introducing the tools and applications. Nat Rev Genet 14, 9–22 (2013). https://doi.org/10.1038/nrg3316
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