Gene machines

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.

Abstract:

Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. In Escherichia coli, UvrA and UvrB initiate NER, although the mechanistic details of how this occurs in vivo remain to be established. Here we provide, using single-molecule fluorescence imaging, a comprehensive characterization of the lesion search, recognition and verification process in living cells. We show that NER initiation involves a two-step mechanism in which UvrA scans the genome and locates DNA damage independently of UvrB. Then UvrA recruits UvrB from solution to the lesion. These steps are coordinated by ATP binding and hydrolysis in the ‘proximal’ and ‘distal’ UvrA ATP-binding sites. We show that initial UvrB-independent damage recognition by UvrA requires ATPase activity in the distal site only. Subsequent UvrB recruitment requires ATP hydrolysis in the proximal site. Finally, UvrA is dissociated from the lesion complex, allowing UvrB to orchestrate the downstream NER reactions.

Abstract:

Herein we investigate the molecular bases of DNA polymerase I conformational dynamics that underlie the replication fidelity of the enzyme. Such fidelity is determined by conformational changes that promote the rejection of incorrect nucleotides before the chemical ligation step. We report a comprehensive atomic resolution study of wild type and mutant enzymes in different bound states and starting from different crystal structures, using extensive molecular dynamics (MD) simulations that cover a total timespan of ~5 ms. The resulting trajectories are examined via a combination of novel methods of internal dynamics and energetics analysis, aimed to reveal the principal molecular determinants for the (de)stabilization of a certain conformational state. Our results show that the presence of fidelity-decreasing mutations or the binding of incorrect nucleotides in ternary complexes tend to favor transitions from closed toward open structures, passing through an ensemble of semi-closed intermediates. The latter ensemble includes the experimentally observed ajar conformation which, consistent with previous experimental observations, emerges as a molecular checkpoint for the selection of the correct nucleotide to incorporate. We discuss the implications of our results for the understanding of the relationships between the structure, dynamics, and function of DNA polymerase I at the atomistic level.

Abstract:

Single-molecule fluorescence and Förster resonance energy transfer (smFRET) are important tools for studying molecular heterogeneity, cellular organization, and protein structure in living cells. However, in vivo smFRET studies are still very challenging, and a standardized approach for robust in vivo smFRET measurements is still missing. Here, we synthesized protected DNAs with chemically linked ends as robust in vivo nano-rulers. We efficiently internalized doubly-labeled end-sealed DNA standards into live bacteria using electroporation and obtained stable and long-lasting smFRET signatures. Single-molecule fluorescence signals could be extended to ∼1 min by studying multi-fluorophore DNA standards. The high stability of protected DNA standards offers a general approach to evaluate single-molecule fluorescence and FRET signals, autofluorescence background, and fluorophore density, and hence, quality check the workflow for studying single-molecule trajectories and conformational dynamics of biomolecules in vivo.

Abstract:

RNA-dependent RNA polymerases (RdRps) are used by RNA viruses to replicate and transcribe their RNA genomes1 . They adopt a closed, right-handed fold with conserved subdomains called palm, fingers and thumb1,2. Conserved RdRp motifs A–F coordinate the viral RNA template, NTPs and magnesium ions to facilitate nucleotide condensation1 . For the initiation of RNA synthesis, most RdRps use either a primer-dependent or de novo mechanism3. The influenza A virus RdRp, in contrast, uses a capped RNA oligonucleotide to initiate transcription, and a combination of terminal and internal de novo initiation for replication4. To understand how the influenza A virus RdRp coordinates these processes, we analysed the function of a thumb subdomain β-hairpin using initiation, elongation and single-molecule Förster resonance energy transfer (sm-FRET) assays. Our data indicate that this β-hairpin is essential for terminal initiation during replication, but not necessary for internal initiation and transcription. Analysis of individual residues in the tip of the β-hairpin shows that PB1 proline 651 is critical for efficient RNA synthesis in vitro and in cell culture. Overall, this work advances our understanding of influenza A virus RNA synthesis and identifies the initiation platform of viral replication.