Beta-Prime Subunit of Bacterial RNA Polymerase

Bacterial RNA Polymerase: New Insights on a Fundamental Molecular MachineBacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine

spinqualitylabelsall models β’ Subunit with Colored Structures 2o5i (See Text) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Abstract

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 aspartate residues, the magnesium ion, the bridge helix, and the trigger helix. The active site channel accommodates double stranded DNA (dwDNA) and an RNA/DNA hybrid. The secondary channel, which is bordered by the rim helices, allows nucleotides (NTPs) to enter the active site. 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. The clamp helices interact with the σ subunit of RNAP.

RNA Polymerase Elongation ComplexRNA 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 (http://www.pingrysmartteam.com/RPo/RPo.htm) (Note: Different nomenclature is used). 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. The β’ subunit contains structures and forms channels that are crucial to this process.

Ribonucleotides enter through the secondary channel (15 x 20 Å). 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. 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 RNAPβ’ Subunit of Thermus thermophilus RNAP

spinqualitylabelsall models Thermus thermophilus β’ Subunit Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

This tutorial describes the of the elongation complex of Thermus thermophilus RNAP. The β’ subunit contains structures and channels required for RNA transcription.

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 provides the complementary sequence for the RNA transcript and continues along the active site channel adjacent to the active site. The , or coding strand, is held away from the active site by the rudder and clamp helices (not shown in the model).

The , which is bordered by the , forms the entrance for NTPs into RNAP. As the NTPs enter, it is proposed that Met1238 and aid in selection and positioning of the cognate NTP, while works to discriminate against non-cognate NTPs by evaluating ribose hydroxyls for hydrogen bonding.

The at the junction between the dwDNA and the RNA/DNA hybrid. The base pair at the is distorted. The on the template strand occurs at the kink.

Upstream of the active site is the . 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 .

The stabilizes the dwDNA and the upstream RNA/DNA hybrid with numerous sidechain interactions. The are shown contacting the dwDNA and RNA/DNA structures. The rudder meets the which interact with the σ subunit of RNAP.

The internal chamber can accommodate the 9 bp . At the upstream position the hybrid meets the that sterically blocks continued elongation of the hybrid. The 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 consists of three highly conserved aspartate sidechains (Asp739, Asp741, Asp743) chelated to the Mg2+ ion required for catalysis. Phosphodiester bond formation that occurs during catalysis involves the , the , and the , 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 LoopNucleotide Addition and the Trigger Loop

This section will feature a morphing animation demonstrating the conformational changes undergone by the trigger loop/helix when switching from the pre-insertion complex to the insertion complex. The animation can currently be found at http://www.molmovdb.org/cgi-bin/morph.cgi?ID=807081-19674 and was designed by Mark Hoelzer of the Center for BioMolecular Modeling at MSOE. The conformational change animation is an interpretation of static models, but does not represent the actual conformational change.

2011 UW-Milwaukee CREST Team2011 UW-Milwaukee CREST Team

Team MembersTeam Members

Catherine L Dornfeld

Christopher Hanna

Jason Slaasted

AcknowledgmentsAcknowledgments

Steven Forst, Ph.D., University of Wisconsin-Milwaukee

Rick Gourse, Ph.D., University of Wisconsin-Madison

MSOE Center for BioMolecular Modeling: Mark Hoelzer, Margaret Franzen, Ph.D. and Tim Herman, Ph.D.

NSF CREST Program