The transcription of RNA from a DNA template is an essential and highly regulated step in every living organism. The multisubunit RNA polymerase (RNAP) enzymes that are required for transcription are conserved throughout evolution and rely on various initiation factors to target RNAP to specific genes in order to tune gene expression to different environments. Bacterial initiation factors are known as σ (sigma) factors and can be grouped into two classes based on their requirement for ATP hydrolysis during initiation.
We have used cryo-electron microscopy to study the individual states required en route to transcription initiation via the σ54 factor which requires ATP hydrolysis by an ATPase activator.
Our data provide insight throughout the σ54 initiation pathway from the point of DNA binding to initial transcription of RNA. Through the analysis of these structures, we propose the fundamental process of DNA unwinding is coupled with its loading into the active site cleft and that this is conserved throughout alternative initiation systems in bacteria, archaea and eukaryotes. We refer to this as a ‘coupled load and unwind’ model.
This work will help develop our understanding of both the fundamental processes involved in initiation in all species and also the specific pathway that many bacteria use to tune gene expression in certain environments.