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Thermophilic DNA Polymerase Alpha-Subunit

Introduction

Role of DNA Polymerase in Replication

HOW MUCH DETAIL TO INCLUDE?

A primary function of DNA polymerase is to replicate, or copy, the DNA of an organism. DNA replication occurs just prior to cell division is necessary for growth and reproduction of a living organism (see DNA Replication, Transcription and Translation). DNA consists of two strands of connected deoxynucleotides (dNTPs) running anti-parallel to each other in a double helix. Deoxynucleotides consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The nitrogen bases are adenine (A), cytosine (C), guanine (G), and thymine (T). The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds. Overall, the backbone has a negative charge. The nitrogen bases of the dNTPs extend into the helix and form pairs that are stabilized by hydrogen bonds. Due to the structure of the nitrogen bases, these hydrogen bonds always form so that adenine pairs with thymine and cytosine pairs with guanine (Watson-Crick base pairs). When the base pairs are formed, double-stranded DNA results.

The replisome is an enzymatic complex with several subunits. There are the core polymerase subunits (α, ε, and θ), the β-clamp, and the clamp loader complex. This tutorial will focus on the α-subunit because it contains the catalytic site for dNTP addition and elongation of the nascent DNA strand.

Image:Http://origin-ars.els-cdn.com/content/image/1-s2.0-S1570963909001897-gr3.jpg

DNA Polymerase III in Bacteria

DNA polymerase III is the primary replicative polymerase in bacteria.

The crystallized structure of Thermus aquaticus (Taq) DNA polymerase III α subunit was the first to contain the ternary complex (polymerase, DNA and incoming dNTP). Taq DNA pol III is homologous to that of Escherichia coli and also Polβ, a eukaryotic non-replicative repair polymerase.

Learning Objectives

• How does the α subunit of DNA polymerase III contact DNA?
• Which domains select for deoxyribonucleotides?
• Which domains form the catalytic site?
• Which domains form the DNA exit channel?

Components of Taq DNA Polymerase III α-Subunit

VIDEO 1 VIDEO 2

Important Domains

The fingers, palm and thumb are highly conserved domains. They are nicknamed as such because the structures resemble a closed hand. They form the nucleotide binding pocket and are involved in nucleotide positioning and sugar selection, nucleotide addition, and DNA interactions.

• Fingers - selects for deoxyribonucleotides, positions incoming dNTPs, and forms part of the active site
• Palm - contains the catalytic site for nucleotide addition and aids incoming nucleotide positioning
• Thumb - forms part of the DNA exit channel and interacts with DNA
• β-binding domain - binds β-sliding clamp and positions double-stranded DNA
• C-terminal domain (CTD) - contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove
• Polymerase and histodinol phosphatase (PHP) domain - forms part of the DNA exit channel and has potential proofreading function via zinc-ion dependent exonuclease activity

DNA Interactions & Conformational Changes

The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA. The β-binding domain, fingers and thumb also adjust.

Nucleotide Addition

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 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].

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