Sigma 54 minds the gap

 A cryo–electron microscopy reconstruction of the RNA polymerase–σ⁵⁴ –PspF–DNA co-complex; promoter DNA is orange. Figure courtesy of D. Bose and X. Xhang, Imperial College, London, UK.

Using cryo–electron microscopy (cryo–EM) and photocrosslinking to capture the initiation of transcription by Escherichia coli RNA polymerase (RNAP)–σ⁵⁴, Bose et al. report in Molecular Cell that σ⁵⁴ can block access of single-stranded DNA to the active site of RNAP. An interaction with an ATP-hydrolysing transcriptional activator removes this block and enables transcription to proceed.

The main bacterial sigma factor (σ⁷⁰) transcribes housekeeping genes, whereas the alternate sigma factor (σ⁵⁴) transcribes a subset of genes. Despite limited amino-acid similarity between σ⁵⁴ and σ⁷⁰, these sigma factors bind to overlapping regions of the RNAP. However, whereas the RNAP–σ⁷⁰ holoenzyme is competent for transcription, the RNAP–σ⁵⁴ complex binds to promoter DNA, then remains stalled and unable to transcribe. When the activator protein PspF (an AAA+ family protein) binds to a bacterial enhancer-like sequence in the promoter and hydrolyses ATP, the RNAP–σ⁵⁴ complex is converted from a 'closed' (bound but stalled) to an 'open' complex that can actively transcribe. Activation of RNAP–σ⁵⁴ transcription by PspF is therefore analogous to activation of RNA polymerase II-mediated transcription in eukaryotes by the ATP-hydrolysing protein TFIIH.

Cryo–EM reconstruction was used to reveal interactions made between RNAP, σ⁵⁴ and the activator protein PspF. Bose et al. compared the cryo–EM reconstruction of RNAP with RNAP–σ⁵⁴, fitted into this the crystal structure of the RNAP enzyme from Thermus thermophilus and imaged RNAP complexed with truncated versions of σ⁵⁴, or a form of σ⁵⁴ tagged at the carboxyl terminus with nanogold, to reveal the position of the C-terminal promoter recognition domain of the sigma factor within the holoenzyme. To identify the likely position of DNA in the RNAP–σ⁵⁴ complex, they inspected cryo–EM reconstructions of RNAP–σ⁵⁴ and T. thermophilus holoenzyme complexed with DNA. Strikingly, the σ⁵⁴ C-terminal promoter interaction domain seems to misalign promoter DNA with the active site of the RNAP.

It was important to locate the region I domain of σ⁵⁴ because this domain not only serves to inhibit transcription in the closed complex but also directly interacts with the transcriptional activator PspF and is necessary for open-complex formation. Comparing a reconstruction of a holoenzyme complex that contained σ⁵⁴ lacking region I with the wild-type holoenzyme showed that region I of σ⁵⁴ occludes DNA from the active site of RNAP, further accounting for the inability of the closed complex of RNAP–σ⁵⁴ to transcribe.

To probe how binding to the activator protein alters the interactions among the components of the transcription complex, Bose et al. analysed a cryo–EM reconstruction of RNAP–σ⁵⁴ –PspF (amino acids 1–275) stabilized with a non-hydrolysable analogue of ATP. This revealed that region I of σ⁵⁴ is rearranged after binding the activator protein PspF and no longer fully blocks the active site of the enzyme. Interaction with PspF also moves σ⁵⁴ domains, which causes the misaligned promoter DNA to slide into the correct position for DNA melting and template-strand loading. By photocrosslinking promoter probes to the holoenzyme complex, they proved that the activator works in front of the RNAP to remodel the complex and activate transcription.

The initiation of bacterial transcription in E. coli serves as a model for transcription in Bacteria, Eukarya and Archaea. This is the first time that structural features of activator-driven domain rearrangements that occur in transcription initiation have been visualized. The next step is to examine the details of the nucleotide-dependent transactions that underlie this model for transcription initiation.