What have single-molecule studies taught us about gene expression?

Abstract

The production of a single mRNA is the result of many sequential steps, from docking of transcription factors to polymerase initiation, elongation, splicing, and, finally, termination. Much of our knowledge about the fundamentals of RNA synthesis and processing come from ensemble in vitro biochemical measurements. Single-molecule approaches are very much in this same reductionist tradition but offer exquisite sensitivity in space and time along with the ability to observe heterogeneous behavior and actually manipulate macromolecules. These techniques can also be applied in vivo, allowing one to address questions in living cells that were previously restricted to reconstituted systems. In this review, we examine the unique insights that single-molecule techniques have yielded on the mechanisms of gene expression.

Keywords: fluorescence; single molecule; splicing; transcription.

Figure 1.

TF target search in an increasingly complex in vivo environment. To reconcile unexpectedly fast TF search times, facilitated diffusion was proposed. In this model, TFs bind unspecifically when they collide with DNA while diffusing within the nucleus and then slide along the DNA until they arrive at the correct site or fall off. At low protein concentrations, sliding is expected to speed up the search times, but at high protein concentrations, this is not expected to be the case. Furthermore, the crowded environment within the nucleus, where DNA-bound obstacles or higher-order chromatin architecture exist, is expected to complicate TF search modes. Reprinted by permission from Macmillan Publishers Ltd.: Nature Structural and Molecular Biology (Wang et al. 2013) © 2013.

Figure 2.

Transcriptional bursting visualized with direct measurement of nascent RNA. (A–D) Time-lapse microscopy of single-copy reporter gene expression in U2OS cells. Nascent RNA was visualized by the binding of the high-affinity MS2 protein to RNA stem loops. (E,F) Integrated fluorescence intensity, which reflects nascent RNA, was plotted as a function of time for two individual genes in two different cells. On and off periods of the transcriptional burst are indicated. Reprinted from Larson et al. 2013.

Figure 3.

EM analysis of PH05 plasmids isolated from S. cerevisiae. The nucleosome position is inferred from the position of single-stranded loops stabilized through psoralen cross-linking. The stochastic partitioning between different nucleosome occupancy states can be directly determined from the electron micrograph. (Left diagram) Structure of the PHO5 gene, with ovals representing nucleosomes. The arrow indicates the transcription start site. Reprinted from Brown et al. 2013.

Figure 4.

Spliceosome assembly follows a highly ordered, reversible pathway. The individual rate constants as determined from single-molecule fluorescence time-lapse recordings of dwell time are indicated. Note the absence of a single rate-limiting step that dominates the kinetics. From Hoskins et al. (2011). Reprinted with permission from AAAS.