Group:SMART:2006 Pingry SMART Team

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RNA Polymerase Holoenzyme - Open Promoter Complex (RPo)

SMART Teams (Students Modeling A Research Topic) are teams of high school students and their teachers who work with research scientists to design and construct physical models of proteins or other molecular structures that are being investigated in the scientists’ laboratories. S.M.A.R.T. Teams use state-of-the-art molecular design software and rapid prototyping technologies to produce these unique models.

With the support of the Center for Biomolecular Modeling at the Milwaukee School of Engineering, Pingry has established S.M.A.R.T. Teams since the 2003-2004 academic year. The 2006 Pingry S.M.A.R.T. Team worked with Seth Darst, Rockefeller University.

Discussions with Seth Darst allowed the Pingry S.M.A.R.T. Team to use RP RasMol to design models of RNA polymerase, highlighting aspects of the enzyme’s function and its interaction with antibiotics. These final designs were used to direct rapid prototyping machines to build physical models. These physical models are “communication tools” that can be used to enhance the understanding of RNA polymerase and the transcription cycle among the scientific and academic community. By contributing this new tool to Seth Darst’s research team, the students have had the opportunity to experience and participate in “real science” as it is practiced in an active research lab.

RNA Polymerase Holoenzyme - Open Promoter Complex (RPo)

RNA polymerase (RNAP) is the enzymatic machinery responsible for transcription, a key regulatory step in gene expression. The prokaryotic RNAP is a highly conserved, "crab claw" shaped enzyme with a molecular mass of ~400kD. In order to recognize a promoter to begin transcription, the 5-subunit core enzyme (α,α,β,β’,ω) must bind to one of various sigma (σ) factors; this form of the enzyme is called the holoenzyme. Each of the different σ factors recognize different promoter elements upstream of genes allowing the cell to respond to various environmental cues. Once holoenzyme binds the promoter, DNA downstream of this interaction is brought into the enzyme and melted to expose single-stranded DNA. This stage of transcription initiation is described here using a model of RNA polymerase-open-promoter-complex (RPo).

Structural characteristics

Core enzyme requires the addition of the σ factor (forming holoenzyme) to be able to recognize the promoter elements. The six base pairs on the -10 region, two on the extended -10 region, and six base pairs on the -35 region of is recognized by domains 2, 3, and 4 of σ factor, respectively.

Promoter sequence can vary in "affinity" for RNA polymerase holoenzyme depending on its deviation from the consensus sequence. Similarly, each σ factor recognizes different promoter sequences. These interactions contribute to gene regulation in prokaryotes by putting various genes under control of separate σ factors. RNAP-promoter interactions are, therefore, key to the control of gene expression.

Between the β and β’ regions of RNAP there is a single alpha helix called the bridging helix. The β and β’ regions make up a “crab claw” shape that is characteristic of this RNAP. The bridging helix connects these two regions, creating two channels. Down-stream DNA enters the larger of these channels where it is split to create the transcription bubble spanning 12-14 nucleotides. The smaller of the two is the secondary channel, where RNA nucleotides enter the complex towards the single stranded template stand near the RNAP active site. With its 12 Å diameter, the secondary channel is only large enough to allow these nucleotides to pass through (the larger diameter of a double-stranded DNA (20 Å) is much too large to pass through the secondary channel). The bridging helix also seems to be supporting the β and β’ regions apart from each other and creating the space in which the RNA nucleotides can reach the active site Mg2+, which is located directly opposite from the helix itself. Notice, when looking directly into the secondary channel, that both Mg2+ ion and single stranded template DNA is visible.

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