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Revision as of 22:08, 25 August 2011

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

This is the β’ subunit of the elongation complex of Thermus thermophilus RNAP. The beta-prime subunit contains structures and channels required for RNA transcription.

DNA in the active site channel provides the genetic information for RNA transcription. The active site channel that accommodates the downstream DNA (dwDNA) and RNA/DNA hybrid is 27 Å wide. The template strand provides the complementary sequence for the RNA transcript and continues along the active site channel adjacent to the active site. The non-template strand, or coding strand, is held away from the active site by the rudder and clamp helices (not shown in the model).

The template strand is kinked at the junction between the dwDNA and the RNA/DNA hybrid. The base pair at the +2 site is distorted. The unpaired acceptor DNA at position +1 on the template strand occurs at the kink.

Upstream of the active site is the RNA/DNA hybrid. This hybrid structure is comprised of the template strand and the complementary RNA transcript hydrogen-bonded to the template bases. The most recently formed hybrid bond is located at the -1 site.

The rudder stabilizes the dwDNA and the upstream RNA/DNA hybrid with numerous sidechain interactions. The side chains of two arginine residues are shown contacting the dwDNA and RNA/DNA structures.

The internal chamber can accommodate the 9 bp RNA/DNA hybrid. At the upstream position the hybrid meets the lid that sterically blocks continued elongation of the hybrid. The lid facilitates cleavage of the H-bond releasing the growing RNA transcript into the exit channel. As the bond is cleaved, the template strand moves one position forward through the active site channel. This process is called translocation. This allows the only unpaired template nucleotide to move into the +1 site adjacent to the active site where nucleotide addition occurs.

The active site consists of three highly conserved aspartate sidechains chelated to the Mg2+ ion required for catalysis. Phosphodiester bond formation that occurs during catalysis involves the active site Mg2+ ion, the bridge helix, and the trigger helix, which is unstructured in this model due to its high mobility. During nucleotide addition, the alpha-phosphate of the incoming ribonucleotide triphosphate (NTP) reacts with the 3’ hydroxyl of the last ribonucleotide in the RNA transcript.

After catalysis the RNA/DNA hybrid moves in the -1 site, and the ribonucleotide in this bond provides the 3’ hydroxyl for the next incoming NTP.

Nucleotide Addition and the Trigger Loop

This section will feature a video explaining the conformational changes undergone by the trigger loop/helix when switching from the pre-insertion complex to the insertion complex.

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

NSF 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