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Thermophilic DNA Polymerase Alpha-Subunit

Introduction

Function of DNA Polymerase

• Replication

DNA consists of two strands of joined deoxynucleotides (dNTPs) running anti-parallel to each other in a double helix. The backbone of the DNA consists of sugar groups and phosphates; these lend a negative charge. Nitrogen bases are connected to the backbone and The nitrogen bases of the dNTPs extend into the helix and form pairs stabilized by hydrogen bonds. Due to the structure of the nitrogen bases, these hydrogen bonds form in very specific ways. As a result,

• Replisome = replication machinery
  • Core polymerase subunits - α, ε, θ
  • β-clamp
  • Clamp loader complex

• Proofreading

DNA Polymerase III in Bacteria

Learning Objectives

Components of Taq DNA Polymerase III Alpha-Subunit

Model features ternary complex - enzyme + DNA + dNTP Homologous to DNA polymerase III alpha subunit of E. coli Homologous to Pol-Beta, a eukaryotic DNA repair polymerase, instead of eukaryotic replicative polymerase

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VIDEO OF PLAYERS

• Histidine kinase (HK) = CheA, found as a dimer
  • P1 = phosphotransfer (His48)
  • P2 = CheY binding
  • P3 = dimerization
  • P4 = ATP kinase
  • P5 = CheW site
• Response regulator (RR) = CheY
  • Asp57 = phosphate accepting site
• Roles of CheB, CheR and CheZ

Phosphotransfer Events

VIDEOS OF PHOSPHORYLATION EVENTS

• Autophosphorylation of CheA at His48
• Phosphotransfer of labile phosphate to CheY at Asp57
• Diffusion of CheY away from CheA and towards FliM at flagellar motor
• Increase in attractant (serine) concentration
  • Serine binds to receptor and blocks auto-phosphorylation of CheA
  • CheY does not receive phosphate
  • Motor resets and results in counterclockwise rotation and running motion

Learning Objectives

• How do you get specific interactions between CheA and CheY?
• What is the role of P1 in the interaction?
• What is the role of P2 in the interaction?
• How do you position His48 and Asp57 to allow phosphotransfer?

Specificity of CheA-CheY Interactions

TUTORIAL/VIDEO

Roles of CheA Domains

Role of P1 in CheA-CheY Interactions

TUTORIAL/VIDEO

• Low affinity binding
• CheA-P1 has low affinity for CheY
• Phe8 and Glu15 play role in correct orientation of components for phosphotransfer. Mutations result in significant decreases in phosphotransfer rate.
• Essential to orientation

Role of P2 in CheA-CheY Interactions

TUTORIAL/VIDEO

• High affinity binding
• CheA-P2 has high affinity for CheY. Phe214 on CheA serves as anchor site. Mutations of Phe214 result in four-fold (?) decrease in binding affinity.
• Essential to docking

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2012 UW-Milwaukee CREST Team

Team Members

Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

Abstract

ABSTRACT

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Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine

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 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 ^[1]. 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)^[2]. 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)^[3]. 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^[3].

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^[3]. 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 ^[3]. 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 ^[3].

β’ Subunit of Thermus thermophilus RNAP

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

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. ↑ ^{3.0 3.1 3.2 3.3 3.4} 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