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==2011 UW-Milwaukee CREST Team== | ==2011 UW-Milwaukee CREST Team== | ||
| + | ===Team=== | ||
Catherine L Dornfeld | Catherine L Dornfeld | ||
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Jason Slaasted | Jason Slaasted | ||
| - | == | + | ===Acknowledgments=== |
| - | + | Steven Forst, Ph.D., University of Wisconsin-Milwaukee | |
| - | + | Rick Gourse, Ph.D., University of Wisconsin-Madison | |
| - | + | MSOE Center for BioMolecular Modeling & the CREST program | |
| + | ==References== | ||
| - | + | Snyder, L & Champness, W (2007). ''Molecular genetics of bacteria'' (3rd ed.). Washington, D.C.: ASM Press. | |
| - | + | ||
| - | + | Vassylyev DG, Vassylyeva MN, Zhang J, Palangat M, Artsimovitch I, Landick R. ''Structural basis for substrate loading in bacterial RNA polymerase''. Nature. 2007 Jul 12;448(7150):163-8. Epub 2007 Jun 20. PMID:17581591 doi:10.1038/nature05931 | |
| - | + | Vassylyev DG, Vassylyeva MN, Perederina A, Tahirov TH, Artsimovitch I. ''Structural basis for transcription elongation by bacterial RNA polymerase''. Nature. 2007 Jul 12;448(7150):157-62. Epub 2007 Jun 20. PMID:17581590 doi:10.1038/nature05932 | |
Revision as of 04:56, 16 August 2011
Contents |
Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine
RNA polymerase (RNAP) is an information-processing molecular machine that copies DNA into RNA. It is a multi-subunit complex found in every living organism. Bacterial RNAP contains six subunits (ββ’α2ωσ). This model focuses on the β’ subunit of RNAP elongation complex (EC) of Thermus thermophilus that contains the active site sequence and several structures involved in the catalytic mechanism. The active site channel accommodates double stranded DNA and an RNA-DNA hybrid helix. The secondary channel allows nucleotides (NTPs) to enter the active site, and the exit channel guides the growing RNA transcript out of the complex. The DNA template strand becomes kinked as it moves through the active site channel and is separated from the non-template strand. This kink allows one dNTP at a time to become available for nucleotide addition once it translocates to the +1 site. The bridge helix (BH) and trigger loop (TL) work together as a “swinging gate” to enhance the catalytic action by facilitating NTP addition. In the crystal structure of the EC without NTP in the active site, the TL (β’ 1236-1265) is unstructured. In the EC crystal structure with a non-hydrolysable nucleotide (AMPcPP), the TL folds into two anti-parallel helices (trigger helix, TH) that interact with the adjacent BH to create a three-helical bundle forming a catalytically active complex. The other structures that are functionally important in the β’ subunit are the “lid” (β’ 525-539) that cleaves the RNA-DNA hybrid, directing the newly formed RNA out through the exit channel, and the “rudder” (β’ 582-602) that helps to stabilize the DNA helix and the RNA-DNA hybrid in the active site channel.
RNA Polymerase Elongation Complex
The RNAP holoenzyme is a molecular machine comprised of six subunits that copies DNA to RNA. RNAP initially binds to DNA at the promoter to form the closed complex. The DNA surrounding the promoter sequence unwinds to form the open complex consisting of a 17 base pair transcription bubble (link to Pingry model with footnote for different nomenclature). The transcribed template strand is held inside the active site channel while the non-template strand is held between the rudder and clamp helices, away from the active site. RNAP releases from the promoter and transitions to the elongation complex that moves along the template strand, adding nucleotides to the 3’ hydroxyl of the RNA at a rate of 30 to 100 nucleotides per second. The β’ subunit contains structures and forms channels that are crucial to this process.
Ribonucleotides enter through the secondary channel (15 x 20 Å)(link to Pingry model). The ribonucleotide is initially positioned at the pre-insertion site with its base forming hydrogen bonds with the template base and the triphosphate facing the active site. Subsequent movement of the ribonucleotide to the insertion site positions the triphosphate close enough to the active site for catalysis to occur.
The active and secondary channels are separated by the bridge helix (link to Pingry model). Besides forming channels, the bridge helix interacts with a structure called the trigger loop, which is unstructured in this model. When a nucleotide is present, the bridge helix induces a conformation change in the trigger loop so it becomes the trigger helix. The trigger helix acts as a swinging gate while guiding ribonucleotides into their correct orientation to meet the 3’ hydroxyl of the growing RNA transcript. The trigger helix also reduces the size of the secondary channel to 11 x 11 Å, which prevents diffusion of the complementary nucleotide away from the active site while simultaneously preventing interference from other nucleotides.
β’ Subunit of Thermus thermophilus RNAP
Insert tutorial here
Nucleotide Addition and the Trigger Loop
Insert video here
2011 UW-Milwaukee CREST Team
Team
Catherine L Dornfeld
Christopher Hanna
Jason Slaasted
Acknowledgments
Steven Forst, Ph.D., University of Wisconsin-Milwaukee
Rick Gourse, Ph.D., University of Wisconsin-Madison
MSOE Center for BioMolecular Modeling & the CREST program
References
Snyder, L & Champness, W (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.
Vassylyev DG, Vassylyeva MN, Zhang J, Palangat M, Artsimovitch I, Landick R. Structural basis for substrate loading in bacterial RNA polymerase. Nature. 2007 Jul 12;448(7150):163-8. Epub 2007 Jun 20. PMID:17581591 doi:10.1038/nature05931
Vassylyev DG, Vassylyeva MN, Perederina A, Tahirov TH, Artsimovitch I. Structural basis for transcription elongation by bacterial RNA polymerase. Nature. 2007 Jul 12;448(7150):157-62. Epub 2007 Jun 20. PMID:17581590 doi:10.1038/nature05932
