Beta-Prime Subunit of Bacterial RNA Polymerase

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1 Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine
- 1.1 Introduction to RNAP
- 1.2 Learning Objectives
2 Tutorial: β’ Subunit of Thermus thermophilus RNAP
- 2.1 Nucleotide Addition Cycle
- 2.2 Challenge Questions
3 2011 UW-Milwaukee CREST Team
4 References

Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine

Introduction to RNAP

RNA polymerase (RNAP) is a molecular machine comprised of six subunits (ββ’α2ωσ) that copies DNA into RNA. RNAP initially binds to DNA at the promoter, forming the closed complex^[1]. The DNA surrounding the promoter sequence unwinds and forms the open complex (http://www.pingrysmartteam.com/RPo/RPo.htm - please note that different nomenclature is used)^[2]. RNAP releases from the promoter and transitions into the elongation complex (EC). The EC moves along the template strand, adding ribonucleotides to the 3’ hydroxyl of the RNA transcript.

The β’ subunit contains structures important to this function, including the sites for ribonucleotide addition to the RNA transcript and catalysis. The bridge helix and trigger helix work together to facilitate ribonucleotide positioning near the active site, while the rudder and lid assist in DNA translocation through RNAP.

Video 1: RNA Polymerase Elongation Complex <swf width="422" height="317">http://myweb.msoe.edu/~hoelzer/CrestUWMilwaukee2011-Video1.swf</swf> Video 2: Elongation & Translocation <swf width="422" height="317">http://myweb.msoe.edu/~hoelzer/CrestUWMilwaukee2011-Video2.swf</swf>

Learning Objectives

Examine the bending of DNA in the active site channel
Determine how ribonucleotides enter the active site
Address how RNA polymerase discriminates between ribonucleotides and deoxyribonucleotides
Describe how ribonucleotide triphosphates (NTPs) are oriented correctly in the active site for catalysis
Evaluate the conformational changes of the trigger loop
Describe how the trigger helix is involved in catalysis

Tutorial: β’ Subunit of Thermus thermophilus RNAP

DNA Translocation and the RNA/DNA Hybrid

in the active site channel provides the genetic information for RNA transcription. The active site channel is 27 Å wide and accommodates both (dwDNA) and an ^[3]. The (blue) provides the complementary sequence for the RNA transcript and threads through the active site channel adjacent to the active site. The (dark blue), or coding strand, is held away from the active site by the clamp helices and rudder.

The template strand is kinked at the junction between the dwDNA and RNA/DNA hybrid^[4]. The lone unpaired acceptor template base at the is located at the kink^[4]. The base pair at the is distorted^[4]. Upstream of the kink is the . This hybrid structure is comprised of the template strand and the complementary RNA transcript connected by hydrogen bonds. The most recently formed hybrid bond is located at the ^[4].

The (coral) stabilizes the dwDNA and upstream RNA/DNA hybrid with numerous sidechain interactions. Two are shown contacting the dwDNA and RNA/DNA hybrid^[4]. The rudder meets the (dark grey) which interact with the σ subunit of RNAP.

The upstream internal chamber can accommodate a 9-bp RNA/DNA hybrid^[4]. The RNA/DNA hybrid encounters the (light green) that sterically blocks continued elongation^[4]. The lid facilitates , releasing the growing RNA transcript into the exit channel. As the bond is cleaved, the template strand moves one position upstream through the active site channel. This process is called translocation. This allows the lone to move into the +1 site adjacent to the active site where nucleotide addition occurs^[4].

Nucleotide Addition

The , which is bordered by the (very dark blue), forms the entrance for both ribonucleotides (NTPs) and deoxyribonucleotides (dNTPs) into RNAP. The secondary channel's dimensions are 15 x 20 Å, preventing the entrance of dsDNA^[5]. Nucleotides are coupled to a magnesium ion (MgII) as they enter. (indigo) evaluates ribose hydroxyl groups in order to discriminate between NTPs and dNTPs^[5]. The hydrogen bonds on the incoming NTP must complement those of the acceptor template base. As the NTPs enter, it is proposed that (cyan and magenta) aid in selection and orientation of the cognate NTP^[5]. Once the cognate NTP is selected, its alpha and gamma phosphates form temporary phosphate contacts with RNAP near the active site^[5]. The nucleotide adopts a relaxed conformation and resists loading into the catalytic position^[5]. This conformation is influenced by the "basic rim gate" consisting of four residues that surround the NTP phosphates^[5]. Two basic rim gate residues, , are shown (light purple).

The (magenta) separates the active and secondary channels while interacting with the (cyan). In the pre-insertion state, the trigger loop has an unstructured conformation^[5]. Loading of the NTP near the active site induces a conformational change in the trigger loop, and it becomes the two-helical trigger helix^[5]. The "swings" into the secondary channel and changes the dimensions of the channel to 11 x 11 Å^[5]. This reduction in size prevents diffusion of NTP away from the active site while simultaneously preventing interference from other nucleotides^[5]. The presence of the trigger helix causes the NTP phosphate contacts to change^[5]. All three phosphates contact RNAP and adopt a more rigid conformation^[5]. The is now ready for catalysis.

Catalysis

The consists of three highly conserved aspartate sidechains (Asp739, Asp741, Asp743) chelated to a Mg2+ ion (MgI) required for catalysis^[5]. (lime green) chelated to the NTP coordinate positioning of the NTP for catalysis^[6]. The of the RNA transcript has nucleophilic activity and attacks the alpha-phosphate of the NTP^[6]. A phosphodiester bond forms between the RNA transcript and the alpha-phosphate, and the beta- and gamma-phosphates leave as a pyrophosphate group^[6].

After catalysis the RNA/DNA hybrid moves in the , and the ribonucleotide in this bond provides the 3’ hydroxyl for the next incoming NTP^[4].

Nucleotide Addition Cycle

Video 3: The Trigger Helix and Nucleotide Addition <swf width="422" height="317">http://myweb.msoe.edu/~hoelzer/CrestUWMilwaukee2011-Video3.swf</swf>

This section will feature an animation demonstrating the process of nucleotide addition.

An animation showing the conformational changes undergone by the trigger loop/helix when switching from the pre-insertion complex to the insertion complex can currently be found at http://www.molmovdb.org/cgi-bin/morph.cgi?ID=807081-19674. This animation 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.

Challenge Questions

Why does DNA experience a 90 degree bend in the active site channel?
What is the function of the magnesium ion in the active site of RNA polymerase? How does it relate to the magnesium ion coupled to the incoming NTP?
What experiments could prove the ribonucleotide discrimination function of β'Asn737?
What experimental evidence could confirm that the trigger loop to trigger helix conformational change is involved in catalysis?

2011 UW-Milwaukee CREST Team

Team Members

Catherine L Dornfeld, Christopher Hanna and Jason Slaasted

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 ions, 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.

Poster

Acknowledgments

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

References

↑ Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.
↑ 2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial
↑ Zhang G, Campbell EA, Minakhin L, Richter C, Severinov K, Darst SA. Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 A resolution. Cell. 1999 Sep 17;98(6):811-24. PMID:10499798
↑ ^4.0 ^4.1 ^4.2 ^4.3 ^4.4 ^4.5 ^4.6 ^4.7 ^4.8 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
↑ ^5.00 ^5.01 ^5.02 ^5.03 ^5.04 ^5.05 ^5.06 ^5.07 ^5.08 ^5.09 ^5.10 ^5.11 ^5.12 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
↑ ^6.0 ^6.1 ^6.2 Nelson, D. L. & Cox, M. M. (2008). Lehninger principles of biochemistry (5th ed.). New York: W. H. Freeman and Company.

Proteopedia Page Contributors and Editors (what is this?)

Catherine L Dornfeld, Angel Herraez, Michal Harel, Mark Hoelzer, Jaime Prilusky

Retrieved from "http://52.214.119.220/wiki/index.php/Beta-Prime_Subunit_of_Bacterial_RNA_Polymerase"

@@ Line 62: / Line 62: @@
 ==2011 UW-Milwaukee CREST Team==
-===2011 CREST 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 ions, 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.
 ===Team Members===
 Catherine L Dornfeld, Christopher Hanna and Jason Slaasted
+===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 ions, 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.
+===Poster===
+[[Image:UWMFinal.jpg|Catherine L Dornfeld| 350px]]
 ===Acknowledgments===