The crystal structure of E. coli RNA polymearse σ70 holoenzyme

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The Journal of Biological Chemistry (JBC) publishes a little booklet of the "best of jbc." The latest copy arrived in the mail a few days ago and it alerted me to a paper published one year ago on the structure of Escherichia coli RNA polymerase σ⁷⁰ holoenzyme (Murikami, 2013).¹

The control of transcription initiation is a very important topic in biochemistry and molecular biology and the events in E. coli are the model for transcription initiation in all other species. We know more about RNA polymerase and promoter sites in E. coli than in any other species.

You can see how it works by reading: How RNA Polymerase Works: The Chemical Reaction , How RNA Polymerase Works: The Topology of the Reaction and the Structure of the Enzyme , and How RNA Polymerase Binds to DNA. Or you can read a good review by Murakami and Darst (2003).

Here's a brief refresher to bring you up to date. E. coli RNA polymerase is a large enzyme consisting of five subunits: two copies of the α subunit and three others (α₂ββ′ω). There are many different transcription regulatory proteins in the cell and each one recognizes and binds to a different type of promoter. Most of them are called "sigma (σ) factors." The most common factor is σ⁷⁰— it's the one that recognizes the traditional TATA box at the transcription initiation site of most E. coli genes.

The transcription activators bind to RNA polymerase to form a "holoenzyme" complex. This complex binds DNA and slides along it until it encounters the specific sequence recognized by the σ factor. This is the promoter region. The next step involves unwinding a bit of DNA to create a transcription bubble. Then the σ factor dissociates and transcription begins.

The new crystal structure shows the location of σ⁷⁰ for the first time. It's a medium-sized protein (M.W. = 70,000) with five domains. The σ2, σ3, σ4 and σNCR domains form a tight interaction with core RNA polymerase but the flexible end of σ⁷⁰ is the N-terminal domain (σ_1.1) that binds weakly to the main groove where DNA interacts with the holoenzyme. This covers the active site of the enzyme where the first few nucleotides will be polymerized. That σ_1.1 domain is shown as a yellow outline in the figure from the paper (below right). The structure of σ_1.1 was difficult to resolve in the electron density map, indicating that it is not tightly bound.

The idea is that the exposed surface of the σ2 and σ3 domains bind to the TATA box and position the promoter DNA in the blue grove labelled "promoter DNA" in the lower image. (The blue surface indicates basically-charged regions that can bind to the negatively-charged DNA molecule.) Note that the orientation of the molecule in the figure from the paper is the reverse of the figure from my book where the promoter region is on the left.

The flexible σ_1.1 domain prevents formation of the open complex unless the rest of the protein has found a true TATA box. When that happens, the σ_1.1 domain probably uncovers the rest of the DNA grove exposing the site for binding "downstream DNA" and formation of the open complex.

Once the open complex forms, the entire σ factor can dissociate and transcription begins.

Into the textbook it goes!

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JBC publishes a lot of papers and I don't have time to scan the table of contents every few weeks. It's easy to miss important papers this way. I rely on others to find them but that doesn't always work. This is a good example.

Murakami, K.S. (2013) X-ray Crystal Structure of Escherichia coli RNA Polymerase σ⁷⁰ Holoenzyme. Journal of Biological Chemistry 288:9126-9134. [doi: 10.1074/jbc.M112.430900]

Murakami, K.S. and Darst, S.A. (2003) Bacterial RNA polymerases: the wholo story. Current Opinion in Structural Biology 13:31-39. [doi: 10.1016/S0959-440X(02)00005-2]