Linking stochastic fluctuations in chromatin structure and gene expression.

The number of mRNA and protein molecules expressed from a single gene molecule fluctuates over time. These fluctuations have been attributed, in part, to the random transitioning of promoters between transcriptionally active and inactive states, causing transcription to occur in bursts. However, the molecular basis of transcriptional bursting remains poorly understood. By electron microscopy of single PHO5 gene molecules from yeast, we show that the "activated" promoter assumes alternative nucleosome configurations at steady state, including the maximally repressive, fully nucleosomal, and the maximally non-repressive, nucleosome-free, configuration. We demonstrate that the observed probabilities of promoter nucleosome configurations are obtained from a simple, intrinsically stochastic process of nucleosome assembly, disassembly, and position-specific sliding; and we show that gene expression and promoter nucleosome configuration can be mechanistically coupled, relating promoter nucleosome dynamics and gene expression fluctuations. Together, our findings suggest a structural basis for transcriptional bursting, and offer new insights into the mechanism of transcriptional regulation and the kinetics of promoter nucleosome transitions.

In vivo role for the chromatin-remodeling enzyme SWI/SNF in the removal of promoter nucleosomes by disassembly rather than sliding.

Analysis of in vivo chromatin remodeling at the PHO5 promoter of yeast led to the conclusion that remodeling removes nucleosomes from the promoter by disassembly rather than sliding away from the promoter. The catalytic activities required for nucleosome disassembly remain unknown. Transcriptional activation of the yeast PHO8 gene was found to depend on the chromatin-remodeling complex SWI/SNF, whereas activation of PHO5 was not. Here, we show that PHO8 gene circles formed in vivo lose nucleosomes upon PHO8 induction, indicative of nucleosome removal by disassembly. Our quantitative analysis of expression noise and chromatin-remodeling data indicates that the dynamics of continual nucleosome removal and reformation at the activated promoters of PHO5 and PHO8 are closely similar. In contrast to PHO5, however, activator-stimulated transcription of PHO8 appears to be limited mostly to the acceleration of promoter nucleosome disassembly with little or no acceleration of promoter transitions following nucleosome disassembly, accounting for the markedly lower expression level of PHO8.

Quantitative analysis of the transcription control mechanism.

Gene transcription requires a sequence of promoter state transitions, including chromatin remodeling, assembly of the transcription machinery, and clearance of the promoter by RNA polymerase. The rate-limiting steps in this sequence are regulated by transcriptional activators that bind at specific promoter elements. As the transition kinetics of individual promoters cannot be observed, the identity of the activator-controlled steps has remained a matter of speculation. In this study, we investigated promoter chromatin structure, and the intrinsic noise of expression over a wide range of expression values for the PHO5 gene of yeast. Interpretation of our results with regard to a stochastic model of promoter chromatin remodeling and gene expression suggests that the regulatory architecture of the gene expression process is measurably reflected in its intrinsic noise profile. Our chromatin structure and noise analyses indicate that the activator of PHO5 transcription stimulates the rates of promoter nucleosome disassembly, and assembly of the transcription machinery after nucleosome removal, but no other rates of the expression process.

The structure of Escherichia coli signal recognition particle revealed by scanning transmission electron microscopy.

Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for Escherichia coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data were fit into the SRP reconstruction, and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP. Fluorescence resonance energy transfer experiments determined interdomain distances that were consistent with our model of SRP. Docking our model onto the bacterial ribosome suggests a mechanism for signal recognition involving interdomain movement of Ffh into and out of the nascent chain exit site and suggests how SRP could interact and/or compete with the ribosome-bound chaperone, trigger factor, for a nascent chain during translation.