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Single-Molecule Studies of Gene Expression

  • Author(s): Hodges, Hamilton Courtney
  • Advisor(s): Bustamante, Carlos
  • et al.
Abstract

During the twentieth century, researchers made significant advances in understanding the biochemical basis for gene expression. In the twenty-first century, the development of single-molecule manipulation techniques allowed researchers for the first time to directly observe the activities of gene expression in real time. In particular, experiments involving single-molecule visualization and manipulation have revealed the processes of gene expression to be stochastic events governed by the physics of the nanoscale.

Our investigation of eukaryotic transcription using single-molecule optical trapping techniques has shown that RNA polymerase II is a type of molecular motor that periodically disengages its DNA substrate and freely diffuses along it, resulting in transient pausing events. The behavior of the polymerase during these pauses has turned out to be critical for understanding how the polymerase transcribes through nucleosomes. In this dissertation, I report that the nucleosome behaves as a fluctuating barrier that locally but dramatically affects the transcription dynamics of the polymerase. The polymerase, rather than actively separating DNA from histones, functions instead as a ratchet that rectifies nucleosomal fluctuations. We also obtained direct evidence that transcription through a nucleosome involves transfer of the core histones behind the transcribing polymerase via a transient DNA loop. This work has significantly addressed how the interplay between polymerase dynamics and nucleosome fluctuations affects the dynamics of gene expression.

Using optical trapping techniques, we also directly observed the process of translation by the E. coli ribosome for the first time. We observed that translation occurs through successive translocation-and-pause cycles. The distribution of pause lengths indicated that at least two rate-determining processes control each pause. Additionally, we have confirmed that each translocation step measures three bases--one codon--and observed that each step occurs in less than 0.1 s. We also observed that translocation and RNA unwinding are strictly coupled ribosomal functions.

The emerging picture is that gene expression arises from the coordinated activities of specific macromolecular motors on their nucleic acid substrates. Our observations of individual transcription and translation events support a detailed physical understanding of gene expression and its regulation.

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