Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding

Abstract

A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex1–3. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12–14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed4–6. Here we present cryo-electron microscopy structures of bacterial RNAP–promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2—universal structural features of RNAP—in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life.