1a. Mechanisms of gene transcription Gene Machine: bacterial RNA polymerase; In vitro transcription studies

Transcription is the first and most important step in gene expression, the information flow from DNA to RNA to proteins. Most of the regulation of gene expression occurs at the level of transcription, which makes this process vital for dissecting basic genetic mechanisms, for understanding diseases due to de-regulation of gene expression, and for building synthetic gene networks and organisms for a variety of applications.

Bacterial transcription is initiated after the machine of transcription, RNA polymerase (RNAP), associates with a σ initiation factor and recognizes the double-stranded DNA of the promoter region of a gene. After several large conformational changes, the RNAP forms a catalytically competent open complex, which contains a transcription bubble with ~14 base-pairs unwound around the transcription start-site (1). In the presence of nucleotides, RNAP starts RNA synthesis; after an abortive initiation phase that involves DNA scrunching, and during which RNAP makes short RNAs that dissociate from the complex (2, 3), RNAP escapes from promoter DNA and proceeds to elongation by forming an elongation complex. Transcriptional elongation continues, occasionally with pauses of variable duration, until termination signals are encountered.

Many mechanisms of transcription, however, even in bacteria, remain unclear. For example, how, does RNAP recognize specific promoter sequences among vast amounts of non-specific DNA? How does the RNAP “melt” the double helix of the promoter DNA to form an open complex? How fast are the initial RNA chains produced, and what elements control the speed of initial transcription?

We address these questions using single-molecule fluorescence techniques, which are well suited for multi-step processes involving transient intermediates and conformational dynamics. For example, using single-molecule FRET with alternating laser excitation, we have studied the dynamics of the open complex and established that DNA dynamics are an important determinant for start-site selection (4); our work has been later confirmed by several high-profile studies.

We have also studied initial transcription on a lac promoter using real-time observations of DNA scrunching on immobilized transcription complexes. Our work revealed to the discovery of a long pause (“initiation pause,” ∼20 s) after synthesis of a 6-mer RNA; such a pause can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA-exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA (5). Current work explores the sequence-dependence and mechanisms of initiation pausing, additional dynamics of the open complex, and RNAP conformational changes at various stages of initiation and elongation.

 

Initial transcription involves a DNA-scrunching phase, during which stable scrunched states were detected; such states are linked to extensive pausing prior to successful elongation of short RNAs.
 

  1. Murakami KS and Darst SA. Bacterial RNA Polymerases: the wholo story. Curr. Opin. in Struct. Biol. 2003, 13:31-39.
  2. Kapanidis AN, Margeat E, Ho SO, Kortkhonjia E, Weiss S, Ebright RH. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science. 2006, 314(5802):1144-7.
  3. Roberts JW et al., RNA Polymerase, a Scrunching Machine. Science. 2006, 314: 1097.
  4. Robb NC, Cordes T, Hwang LC, Gryte K, Duchi D, Craggs TD, Santoso Y, Weiss S, Ebright RH and Kapanidis AN. The transcription bubble of the RNA polymerase–promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection. Journal of Molecular Biology. 2013, 425(5): 875-885.
  5. Duchi D, Bauer DLV, Fernandez L, Evans G, Robb NC, Hwang LC, Gryte K et al. RNA polymerase pausing during initial transcription. Molecular Cell. 2016, 63(6): 939-950.
List of site pages