Single-molecule analysis of the influenza virus replication initiation mechanism
Biophysical Journal Biophysical Society 114:3 (2018) 246A-246A
Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes
Nucleic Acids Research Oxford University Press 46:2 (2017) 677-688
Abstract:
Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA
Nucleic Acids Research Oxford University Press (2016)
Abstract:
Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3' terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3' terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3' cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3' termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3' terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3' vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provides insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.RNA polymerase pausing during initial transcription
Molecular cell Cell Press 63:6 (2016) 939-950
Abstract:
In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses 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.The role of the priming loop in influenza A virus RNA synthesis
Nature Microbiology Nature (2016)