Stephanie Johnson

Rosa's roses : reduced vowels in American English

Beginning phonetics students are taught that some varieties of American English have two contrasting reduced vowels, transcribed as [e] and [i], illustrated by the unstressed vowels in the minimal pair Rosas vs. roses (e.g. Ladefoged 2001, 2005). However, little seems to be known about the precise nature or distribution of these vowels. This study explores these questions through acoustic analysis of reduced vowels in the speech of nine American English speakers. The results show that there is a fundamental distinction between the mid central [e] vowel that can occur in unstressed word-final position (e.g. in Rosa ), and high reduced vowels that occur in most other unstressed positions, and might be transcribed as [i]. The contrast between pairs like Rosas and roses derives from this difference because the word-final [e] is preserved when an inflectional suffix is added, so the schwa of Rosas is similar to the final vowel of Rosa , whereas the unstressed vowel of roses is the high [i] reduced vowel quality found elsewhere. So the standard transcription of the reduced vowel contrast is justified, but the widespread use of [e] to transcribe word-internal reduced vowels is misleading – mid reduced vowels are generally only found in stem-final position.

Sequence dependence of transcription factor-mediated DNA looping

DNA is subject to large deformations in a wide range of biological processes. Two key examples illustrate how such deformations influence the readout of the genetic information: the sequestering of eukaryotic genes by nucleosomes and DNA looping in transcriptional regulation in both prokaryotes and eukaryotes. These kinds of regulatory problems are now becoming amenable to systematic quantitative dissection with a powerful dialogue between theory and experiment. Here, we use a single-molecule experiment in conjunction with a statistical mechanical model to test quantitative predictions for the behavior of DNA looping at short length scales and to determine how DNA sequence affects looping at these lengths. We calculate and measure how such looping depends upon four key biological parameters: the strength of the transcription factor binding sites, the concentration of the transcription factor, and the length and sequence of the DNA loop. Our studies lead to the surprising insight that sequences that are thought to be especially favorable for nucleosome formation because of high flexibility lead to no systematically detectable effect of sequence on looping, and begin to provide a picture of the distinctions between the short length scale mechanics of nucleosome formation and looping.

Poly(dA:dT)-Rich DNAs Are Highly Flexible in the Context of DNA Looping

Large-scale DNA deformation is ubiquitous in transcriptional regulation in prokaryotes and eukaryotes alike. Though much is known about how transcription factors and constellations of binding sites dictate where and how gene regulation will occur, less is known about the role played by the intervening DNA. In this work we explore the effect of sequence flexibility on transcription factor-mediated DNA looping, by drawing on sequences identified in nucleosome formation and ligase-mediated cyclization assays as being especially favorable for or resistant to large deformations. We examine a poly(dA:dT)-rich, nucleosome-repelling sequence that is often thought to belong to a class of highly inflexible DNAs; two strong nucleosome positioning sequences that share a set of particular sequence features common to nucleosome-preferring DNAs; and a CG-rich sequence representative of high G+C-content genomic regions that correlate with high nucleosome occupancy in vivo. To measure the flexibility of these sequences in the context of DNA looping, we combine the in vitro single-molecule tethered particle motion assay, a canonical looping protein, and a statistical mechanical model that allows us to quantitatively relate the looping probability to the looping free energy. We show that, in contrast to the case of nucleosome occupancy, G+C content does not positively correlate with looping probability, and that despite sharing sequence features that are thought to determine nucleosome affinity, the two strong nucleosome positioning sequences behave markedly dissimilarly in the context of looping. Most surprisingly, the poly(dA:dT)-rich DNA that is often characterized as highly inflexible in fact exhibits one of the highest propensities for looping that we have measured. These results argue for a need to revisit our understanding of the mechanical properties of DNA in a way that will provide a basis for understanding DNA deformation over the entire range of biologically relevant scenarios that are impacted by DNA deformability.

Mechanisms of ATP-Dependent Chromatin Remodeling Motors

Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses.

Multiple LacI-mediated loops revealed by Bayesian statistics and tethered particle motion

The bacterial transcription factor LacI loops DNA by binding to two separate locations on the DNA simultaneously. Despite being one of the best-studied model systems for transcriptional regulation, the number and conformations of loop structures accessible to LacI remain unclear, though the importance of multiple coexisting loops has been implicated in interactions between LacI and other cellular regulators of gene expression. To probe this issue, we have developed a new analysis method for tethered particle motion, a versatile and commonly used in vitro single-molecule technique. Our method, vbTPM, performs variational Bayesian inference in hidden Markov models. It learns the number of distinct states (i.e. DNA–protein conformations) directly from tethered particle motion data with better resolution than existing methods, while easily correcting for common experimental artifacts. Studying short (roughly 100 bp) LacI-mediated loops, we provide evidence for three distinct loop structures, more than previously reported in single-molecule studies. Moreover, our results confirm that changes in LacI conformation and DNA-binding topology both contribute to the repertoire of LacI-mediated loops formed in vitro, and provide qualitatively new input for models of looping and transcriptional regulation. We expect vbTPM to be broadly useful for probing complex protein–nucleic acid interactions.

DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation

As the chief informational molecule of life, DNA is subject to extensive physical manipulations. The energy required to deform double-helical DNA depends on sequence, and this mechanical code of DNA influences gene regulation, such as through nucleosome positioning. Here we examine the sequence-dependent flexibility of DNA in bacterial transcription factor-mediated looping, a context for which the role of sequence remains poorly understood. Using a suite of synthetic constructs repressed by the Lac repressor and two well-known sequences that show large flexibility differences in vitro, we make precise statistical mechanical predictions as to how DNA sequence influences loop formation and test these predictions using in vivo transcription and in vitro single-molecule assays. Surprisingly, sequence-dependent flexibility does not affect in vivo gene regulation. By theoretically and experimentally quantifying the relative contributions of sequence and the DNA-bending protein HU to DNA mechanical properties, we reveal that bending by HU dominates DNA mechanics and masks intrinsic sequence-dependent flexibility. Such a quantitative understanding of how mechanical regulatory information is encoded in the genome will be a key step towards a predictive understanding of gene regulation at single-base pair resolution.

Modulation of DNA loop lifetimes by the free energy of loop formation

Significance Storage and retrieval of the genetic information in cells is a dynamic process that requires the DNA to undergo dramatic structural rearrangements. One prominent but still incompletely understood example of such rearrangements is protein-mediated DNA looping. Here, we use a single-molecule biophysical technique to show that the elasticity of the DNA and protein affects the kinetics of both loop formation and, unexpectedly, loop breakdown, and we develop a theory based on polymer physics to explain the origin of these observations. Our results demonstrate a previously unidentified way to probe the looping reaction pathway and quantify the effects of both the DNA and protein elasticity in looping kinetics, and therefore better understand their roles in many gene-regulatory processes. Storage and retrieval of the genetic information in cells is a dynamic process that requires the DNA to undergo dramatic structural rearrangements. DNA looping is a prominent example of such a structural rearrangement that is essential for transcriptional regulation in both prokaryotes and eukaryotes, and the speed of such regulations affects the fitness of individuals. Here, we examine the in vitro looping dynamics of the classic Lac repressor gene-regulatory motif. We show that both loop association and loop dissociation at the DNA-repressor junctions depend on the elastic deformation of the DNA and protein, and that both looping and unlooping rates approximately scale with the looping J factor, which reflects the system’s deformation free energy. We explain this observation by transition state theory and model the DNA–protein complex as an effective worm-like chain with twist. We introduce a finite protein–DNA binding interaction length, in competition with the characteristic DNA deformation length scale, as the physical origin of the previously unidentified loop dissociation dynamics observed here, and discuss the robustness of this behavior to perturbations in several polymer parameters.

The nucleosomal acidic patch relieves auto-inhibition by the ISWI remodeler SNF2h

ISWI family chromatin remodeling motors use sophisticated autoinhibition mechanisms to control nucleosome sliding. Yet how the different autoinhibitory domains are regulated is not well understood. Here we show that an acidic patch formed by histones H2A and H2B of the nucleosome relieves the autoinhibition imposed by the AutoN and the NegC regions of the human ISWI remodeler SNF2h. Further, by single molecule FRET we show that the acidic patch helps control the distance travelled per translocation event. We propose a model in which the acidic patch activates SNF2h by providing a landing pad for the NegC and AutoN auto-inhibitory domains. Interestingly, the INO80 complex is also strongly dependent on the acidic patch for nucleosome sliding, indicating that this substrate feature can regulate remodeling enzymes with substantially different mechanisms. We therefore hypothesize that regulating access to the acidic patch of the nucleosome plays a key role in coordinating the activities of different remodelers in the cell.