Beta-Prime Subunit of Bacterial RNA Polymerase

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This page, as it appeared on May 30, 2012, was featured in this article in the Journal of Biochemistry and Molecular Biology Education.

Contents

Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine

Introduction to RNAP

RNA polymerase (RNAP) is a molecular machine that copies DNA into RNA and is found in every living organism. The bacterial RNAP complex consists of six subunits (ββ’α2ωσ) and three channels. 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 growing RNA transcript.

This tutorial uses the β’ subunit of the RNAP elongation complex of Thermus thermophilus. The β’ subunit contains structures crucial for transcription, including the sites for ribonucleotide addition and catalysis. Double-stranded DNA enters RNAP through the active site channel, while ribonucleotides (NTPs) enter through the secondary channel. As the downstream DNA (dwDNA) enters, it separates into the template and non-template strands. The template strand forms an approximately 90 degree kink in the active site channel. At the kink, one DNA base pair becomes available for NTP pairing and translocates to the +1 site. An NTP enters the active site and induces conformational change of the trigger loop into the trigger helix. The trigger helix forms a three-helical bundle with the bridge helix. This bundle changes dimensions of the active site and facilitates positioning of the NTP for addition to the growing RNA strand. Upon addition of the nucleotide, the dwDNA and RNA/DNA hybrid translocate through RNAP with stabilization from the rudder. The growing RNA strand is separated by the lid and exits RNAP through the exit channel. The DNA template strand rejoins the non-template strand as it exits the active site channel.



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

Nucleotide Addition Cycle


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 are the functions of the magnesium ion in the active site and the magnesium ion coupled to the incoming NTP? What are they coordinating?
  • 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

Catherine L Dornfeld

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

  1. Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.
  2. 2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial
  3. 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. 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. 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. 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

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