An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts. Significance Statement: The phage nucleus is an enclosed replication compartment built by Chimalliviridae phages that, similar to the eukaryotic nucleus, separates transcription from translation and selectively imports certain proteins. This allows the phage to concentrate proteins required for DNA replication and transcription while excluding DNA-targeting host defense proteins. However, the mechanism of selective trafficking into the phage nucleus is currently unknown. Here we determine the region of a phage nuclear protein that targets it for nuclear import and identify a conserved, essential nuclear shell-associated protein that plays a key role in this process. This work provides the first mechanistic model of selective import into the phage nucleus.

RNA polymerase sliding on DNA can couple the transcription of nearby bacterial operons.

DNA transcription initiates after an RNA polymerase (RNAP) molecule binds to the promoter of a gene. In bacteria, the canonical picture is that RNAP comes from the cytoplasmic pool of freely diffusing RNAP molecules. Recent experiments suggest the possible existence of a separate pool of polymerases, competent for initiation, which freely slide on the DNA after having terminated one round of transcription. Promoter-dependent transcription reinitiation from this pool of posttermination RNAP may lead to coupled initiation at nearby operons, but it is unclear whether this can occur over the distance and timescales needed for it to function widely on a bacterial genome in vivo. Here, we mathematically model the hypothesized reinitiation mechanism as a diffusion-to-capture process and compute the distances over which significant interoperon coupling can occur and the time required. These quantities depend on molecular association and dissociation rate constants between DNA, RNAP, and the transcription initiation factor σ70; we measure these rate constants using single-molecule experiments in vitro. Our combined theory/experimental results demonstrate that efficient coupling can occur at physiologically relevant σ70 concentrations and on timescales appropriate for transcript synthesis. Coupling is efficient over terminator-promoter distances up to ∼1,000 bp, which includes the majority of terminator-promoter nearest neighbor pairs in the Escherichia coli genome. The results suggest a generalized mechanism that couples the transcription of nearby operons and breaks the paradigm that each binding of RNAP to DNA can produce at most one messenger RNA.

Recycling of bacterial RNA polymerase by the Swi2/Snf2 ATPase RapA.

Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in the cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryotic Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so are unclear. We used multiwavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the Escherichia coli transcription cycle. In our experiments, RapA at <5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post termination complex (PTC)-consisting of core RNA polymerase (RNAP)-bound sequence nonspecifically to double-stranded DNA-and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription reinitiation in proteobacterial genomes.

Recycling of Bacterial RNA Polymerase by the Swi2/Snf2 ATPase RapA.

Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryote Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so is unclear. We used multi-wavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the E. coli transcription cycle. In our experiments, RapA at < 5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post-termination complex (PTC) -- consisting of core RNA polymerase (RNAP) bound to dsDNA -- and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription re-initiation in proteobacterial genomes. SIGNIFICANCE: RNA synthesis is an essential conduit of genetic information in all organisms. After transcribing an RNA, the bacterial RNA polymerase (RNAP) must be reused to make subsequent RNAs, but the steps that enable RNAP reuse are unclear. We directly observed the dynamics of individual molecules of fluorescently labeled RNAP and the enzyme RapA as they colocalized with DNA during and after RNA synthesis. Our studies show that RapA uses ATP hydrolysis to remove RNAP from DNA after the RNA is released from RNAP and reveal essential features of the mechanism by which this removal occurs. These studies fill in key missing pieces in our current understanding of the events that occur after RNA is released and that enable RNAP reuse.

RNA polymerase sliding on DNA can couple the transcription of nearby bacterial operons.

DNA transcription initiates after an RNA polymerase (RNAP) molecule binds to the promoter of a gene. In bacteria, the canonical picture is that RNAP comes from the cytoplasmic pool of freely diffusing RNAP molecules. Recent experiments suggest the possible existence of a separate pool of polymerases, competent for initiation, which freely slide on the DNA after having terminated one round of transcription. Promoter-dependent transcription reinitiation from this pool of post-termination RNAP may lead to coupled initiation at nearby operons, but it is unclear whether this can occur over the distance- and time-scales needed for it to function widely on a bacterial genome in vivo. Here, we mathematically model the hypothesized reinitiation mechanism as a diffusion-to-capture process and compute the distances over which significant inter-operon coupling can occur and the time required. These quantities depend on previously uncharacterized molecular association and dissociation rate constants between DNA, RNAP and the transcription initiation factor σ 70 ; we measure these rate constants using single-molecule experiments in vitro. Our combined theory/experimental results demonstrate that efficient coupling can occur at physiologically relevant σ 70 concentrations and on timescales appropriate for transcript synthesis. Coupling is efficient over terminator-promoter distances up to ∼ 1, 000 bp, which includes the majority of terminator-promoter nearest neighbor pairs in the E. coli genome. The results suggest a generalized mechanism that couples the transcription of nearby operons and breaks the paradigm that each binding of RNAP to DNA can produce at most one messenger RNA. SIGNIFICANCE STATEMENT: After transcribing an operon, a bacterial RNA polymerase can stay bound to DNA, slide along it, and reini-tiate transcription of the same or a different operon. Quantitative single-molecule biophysics experiments combined with mathematical theory demonstrate that this reinitiation process can be quick and efficient over gene spacings typical of a bacterial genome. Reinitiation may provide a mechanism to orchestrate the transcriptional activities of groups of nearby operons.

Mechanisms of Scaffold-Mediated Microcompartment Assembly and Size Control.

This article describes a theoretical and computational study of the dynamical assembly of a protein shell around a complex consisting of many cargo molecules and long, flexible scaffold molecules. Our study is motivated by bacterial microcompartments, which are proteinaceous organelles that assemble around a condensed droplet of enzymes and reactants. As in many examples of cytoplasmic liquid-liquid phase separation, condensation of the microcompartment interior cargo is driven by flexible scaffold proteins that have weak multivalent interactions with the cargo. Our results predict that the shell size, amount of encapsulated cargo, and assembly pathways depend sensitively on properties of the scaffold, including its length and valency of scaffold-cargo interactions. Moreover, the ability of self-assembling protein shells to change their size to accommodate scaffold molecules of different lengths depends crucially on whether the spontaneous curvature radius of the protein shell is smaller or larger than a characteristic elastic length scale of the shell. Beyond natural microcompartments, these results have important implications for synthetic biology efforts to target alternative molecules for encapsulation by microcompartments or viral shells. More broadly, the results elucidate how cells exploit coupling between self-assembly and liquid-liquid phase separation to organize their interiors.