In a previous post, I reviewed the kinetics of transcription initiation from the binding of RNAP to the formation of the open complex. In the original formalization by McClure, there was only a single step from RPc to RPo, but subsequent work has identified several other intermediate steps and structures. These will become necessary to consider when we start to look at specific promoters and their regulation. For now, I want to add some perspective on the process by looking at the structures of the closed and open complexes.
The initial step in initiation is the formation of the closed complex. Here, the holoenzyme (core + sigma) binds specifically to a promoter by interaction between the sigma factor and the -10 and -35 regions of the promoter DNA. The structure is visualized in Fig. 1, with a reminder that the DNA double helix extends outwards in both directions.
Figure 1. Holoenzyme interaction with promoter DNA. The -10 and -35 promoter regions are highlighted in yellow. The core enzyme is colored in blue-gray and the sigma factor (sigma 70) in orange. Note that only the backbones of the DNA and protein are shown.
The isomerization step from this structure to the open complex is quite dramatic. A region of ~20 bp is unwound, from just upstream of the -10 region to about 10 bp downstream of the +1 initiation site. Additionally, the template strand is brought to the active site which lies at the base of a cleft in the core enzyme (Fig. 2).
Figure 2. Top view of core-RNA polymerase. The location of the active site is indicated. Notice the very deep cleft formed between the β and β’ subunits.
Figure 3. Side view of core RNA polymerase. Labeling as in Fig. 2.
Structure of the DNA in the open complex
Before getting into the contributions of the protein components of RPo, let’s take a look at the structure of the DNA inside the closed complex in Fig. 4. The structures here were determined by Hudson et al., and the structure can be accessed from the PDB web site. This is only part of a larger structure that I will be building in future posts.
Figure 4. Two views of the DNA in the open complex. The -10 and -35 regions are colored in green; the unwound DNA is colored in yellow, and the base complementary to the +1 site of the RNA is colored in red. The surface of the bound RNAP is represented by green points.
Approximately 22 bp are unwound in this isomerization step. For this reaction to be thermodynamically favorable, new bonds must form between the holoenzyme and the unwound DNA as well as the downstream wound region. Not all of these new bonds need be specific: interactions between the DNA backbone and holoenzyme can make up a substantial portion of the binding energy. Any specific interactions between the holoenzyme and the bases would certainly contribute to stability of RPo.
In the next set of images in Fig. 5, I have included sigma70.
Figure 5. Structure of the open complex: DNA & sigma70. As in Fig. 4, except sigma70 is now shown in space orange.
In Fig. 5A, the close the binding of sigma70(region 4) to the -35 region is clearly seen, and in Fig. 5B, the interaction of sigma70(region 2) with the -10 region is readily seen. Notice the close interactions between the unwound -10 DNA and sigma70(region 2). As mentioned above, the extent of specific and non-specific interactions will likely affect the rate of open complex formation, as well as its stability.
Next up: the up element.