Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 are well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. Phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO-complex formation and provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO-complex formation.

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.

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.

Aggregates of human islet amyloid polypeptide (IAPP) in the pancreas of patients with type 2 diabetes (T2D) are thought to contribute to β cell dysfunction and death. To understand how IAPP harms cells and how this might be overcome, we created a yeast model of IAPP toxicity. Ste24, an evolutionarily conserved protease that was recently reported to degrade peptides stuck within the translocon between the cytoplasm and the endoplasmic reticulum, was the strongest suppressor of IAPP toxicity. By testing variants of the human homolog, ZMPSTE24, with varying activity levels, the rescue of IAPP toxicity proved to be directly proportional to the declogging efficiency. Clinically relevant ZMPSTE24 variants identified in the largest database of exomes sequences derived from T2D patients were characterized using the yeast model, revealing 14 partial loss-of-function variants, which were enriched among diabetes patients over 2-fold. Thus, clogging of the translocon by IAPP oligomers may contribute to β cell failure.

Biological systems are spatially organized. This microscopic heterogeneity has been shown to produce emergent complex behaviors such as bistability. Even though the connection between spatiality and dynamic response is essential to understand biological output, its robustness and extent has not been sufficiently explored. This work focuses on a previously described system which is composed of two monostable modules acting on different cellular compartments and sharing species through linear shuttling reactions. One of the two main purposes of this paper is to quantify the frequency of occurrence of bistability throughout the parameter space and to identify which parameters and in which value ranges control the emergence and the properties of bistability. We found that a very small fraction of the sampled parameter space produced a bistable response. Most importantly, shuttling parameters were among the most influential ones to control this property. The other goal of this paper is to simplify the same system as much as possible without losing compartment-induced bistability. This procedure provided a simplified model that still connects two monostable systems by a reduced set of linear shuttling reactions that circulates all the species around the two compartments. Bistable systems are one of the main building blocks of more complex behaviors such as oscillations, memory, and digitalization. Therefore, we expect that the proposed minimal system provides insight into how these behaviors can arise from compartmentalization.

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.