The physical forces that drive oligomerization of soluble proteins are well understood and have been extensively studied.  For proteins with transmembrane domains — transport enzymes, for example — oligomerization is often essential for function but its physical basis is less clear.  In this project,  Janice Robertson devised a new method based on liposome extrusion and single-molecule fluorescence photobleaching analysis to accurately measure the dimer association free energy of a ClC-type chloride ion/hydrogen ion antiporter.  (Janice started this work when she was a postdoc in Chris Miller’s lab at Brandeis and later completed the project in her own  lab at the University of Iowa.)  The study reveals that ClC-ec1 “is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.”

“In all kingdoms of life, gene transcription is not carried out by RNA polymerase enzymes alone.” Instead, accessory proteins ride along with RNA polymerase molecules as the latter move along a gene, regulating their biological function and controlling gene expression. However, in no cases is the kinetic mechanism of such elongation regulation quantitatively understood.

Sigma proteins are known to be regulators of bacterial transcription initiation. However, previous work suggested that σ⁷⁰ is present on some transcription elongation complexes, although the extent to which it is retained from initiation, how long it remains attached, and its consequences for transcription regulation were unclear. In this study, Tim Harden and his collaborators used a novel multi-wavelength single-molecule fluorescence microscopy approach to directly observe and quantitatively characterize the dynamic interactions of the σ⁷⁰ protein with bacterial RNA polymerase molecules in vitro during active RNA synthesis. Harden is a Brandeis Physics Ph.D. student who is jointly advised by Jeff Gelles and Jane Kondev.  The study demonstrates by direct observation that actively elongating polymerase molecules can retain σ⁷⁰ from initiation into the elongation phase of transcription; shows that retained σ⁷⁰ subunits dissociate so slowly that most are still present on the elongation complex at the end of a long gene; and proves that only the subpopulation of elongating polymerases with bound σ⁷⁰ recognize a class of transcriptional pause sequences which in some contexts play a well-established role in regulating gene expression.

More generally, this study provides the first quantitative framework that defines the post-initiation roles of σ⁷⁰, information that is essential to the understanding of global transcription regulation in bacteria. Furthermore, the work demonstrates a general method for elucidating the dynamic interactions of transcription factors with active elongation complexes; this method has broad application in both prokaryotic and eukaryotic transcription biology.

Resources: Plasmids described in this article are available from Addgene.

[ensemblevideo contentid=Z33TDbsofEW7ofOXsSti8w autoplay=true]Regulation of actin filament length is a central process by which eukaryotic cells control the shape, architecture, and dynamics of their actin networks. This regulation plays a fundamental role in cell motility, morphogenesis, and a host of processes specific to particular cell types. This paper by recently graduated Ph.D. student Jeffrey Bombardier and collaborators resolves the long-standing mystery of how formins and capping protein work in concert and antagonistically to control actin filament length. Bombardier used the CoSMoS multi-wavelength single-molecule fluorescence microscopy technique to to discover and characterize a novel tripartite complex formed by a formin, capping protein, and the actin filament barbed end. Quantitative analysis of the kinetic mechanism showed that this complex is the essential intermediate and decision point in converting a growing formin-bound filament into a static capping protein-bound filament, and the reverse. Interestingly, the authors show that “mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex.” The results define the essential features of the molecular mechanism of filament length regulation by formin and capping protein; this mechanism predicts several new ways by which cells are likely to couple upstream regulatory inputs to filament length control.

Resources: The capping protein expression plasmid described in this article is available from Addgene.

Readers interested in this subject should also see a related article by Shekhar et al published simultaneously in the same journal.  We are grateful to the authors of that article for coordinating submission so that the two articles were published together.