Three-dimensional structure of Escherichia coli RNA polymerase holoenzyme determined by electron crystallography

During transcription in E. coli, the DNA-dependent RNA polymerase locates specific promoter sequences in the DNA template, melts a small region containing the transcription start site, initiates RNA synthesis, processively elongates the transcript, and finally terminates and releases the RNA product. Each step is regulated by interactions between the polymerase, the DNA, the nascent RNA, and a variety of regulatory proteins and ligands. The E. coli enzyme contains a catalytic core of two alpha-subunits, one beta- and one beta'-subunit, with relative molecular masses (Mr) of 36,512, 150,619 and 155,162, respectively. The holoenzyme has an additional regulatory subunit, normally sigma, of Mr 70,236. Preparations may also contain the omega-subunit (Mr approximately 10,000), which can be removed without affecting any known properties of the enzyme. Because the amino-acid sequences of the beta- and beta'-subunits are homologous to those of the largest subunits of the yeast, Drosophila and murine RNA polymerases, it seems likely that essential features of the three-dimensional structure and catalytic mechanism of RNA polymerase are also conserved across species. Crystals of RNA polymerase suitable for X-ray analysis have not yet been obtained, but two-dimensional crystals of E. coli RNA polymerase holoenzyme can be grown on positively charged lipid layers. Electron microscopy of these crystals in negative stain shows the enzyme in projection as an irregularly shaped complex approximately 100 x 100 x 160 A in size. We have now determined the three-dimensional structure by electron microscopy of negatively stained, two-dimensional crystals tilted at various angles to the incident electron beam. We find a structure in RNA polymerase similar to the active-site cleft of DNA polymerase I. In the light of functional similarities between these two enzymes, together with other evidence, this probably identifies the active-site region of RNA polymerase.