“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.