User:Catherine L Dornfeld/Sandbox 1
From Proteopedia
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m (→Thermophilic DNA Polymerase Alpha-Subunit) |
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- | = | + | =Alpha Subunit of ''Thermus aquaticus'' DNA Polymerase III= |
==Introduction== | ==Introduction== | ||
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Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />. | Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication<ref name='eukaryote'>Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm. </ref>. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated <ref name='eukaryote' />. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear<ref name='eukaryote' />. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far<ref name='eukaryote' />. | ||
- | Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex). Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication. | + | Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs <ref name='eukaryote' />. [http://en.wikipedia.org/wiki/DNA_polymerase_III DNA polymerase III] is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of ''Thermus aquaticus'', commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)<ref name='wing'>PMID: 18691598</ref>. Taq DNA polymerase III, a replicative polymerase, is homologous to that of ''Escherichia coli'' and also Polβ, a eukaryotic polymerase specializing in repair instead of replication <ref name='wing' />. |
===Learning Objectives=== | ===Learning Objectives=== | ||
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===Important Domains=== | ===Important Domains=== | ||
- | <Structure load='3e0d' size=' | + | <Structure load='3e0d' size='400' frame='true' align='right' caption='DNA Pol III Alpha Subunit' scene='46/461391/Simple_scene/7' /> |
- | The fingers, palm and thumb domains of DNA polymerase III are highly conserved. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. These domains form binding pockets for both DNA and incoming dNTPs. They are also involved in correct dNTP selection, position and addition along with DNA interactions. | + | The fingers, palm and thumb domains of DNA polymerase III are highly conserved <ref name='kohlstaedt'>PMID: 1377403</ref>. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. These domains form binding pockets for both DNA and incoming dNTPs<ref name='wing' />. They are also involved in correct dNTP selection, position and addition along with DNA interactions <ref name='wing' />. |
- | * Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including | + | * Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved catalytic aspartates<ref name='wing' />. The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates)<ref name='wing' />. |
- | * Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis. The fingers domain | + | * Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group<ref name='wing' />. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis<ref name='wing' />. The fingers domain forms part of the active site<ref name='wing' />. |
- | * Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA. | + | * Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA<ref name='wing' />. |
- | * β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA. | + | * β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA<ref name='wing' />. |
- | * C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove. | + | * C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove<ref name='wing' />. |
- | * Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined. | + | * Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel<ref name='wing' />. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined<ref name='wing' />. |
- | * Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain. | + | * Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain<ref name='wing' />. |
===DNA Interactions & Conformational Changes=== | ===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. | + | The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove<ref name='wing' />. 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<ref name='wing' />. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA<ref name='wing' />. The β-binding domain, fingers and thumb also adjust<ref name='wing' />. |
==Nucleotide Addition== | ==Nucleotide Addition== | ||
- | <StructureSection load='3e0d' size=' | + | <StructureSection load='3e0d' size='400' side='left' caption='DNA polymerase III α-subunit (PDB entry [[3e0d]])' scene='46/461391/Simple_scene/7'> |
[http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition] | [http://cbm.msoe.edu/markMyweb/crestVideos/CrestUWMilwaukee2012-Video3.html Video of Nucleotide Addition] | ||
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===Abstract=== | ===Abstract=== | ||
ABSTRACT | ABSTRACT | ||
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- | ==Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine== | ||
- | <Structure load='2o5i' size='400' frame='true' align='right' caption='β’ Subunit with Colored Structures [[2o5i]] (See Text)' scene='User:Catherine_L_Dornfeld/Sandbox_1/Beta_prime_1_spin/1' /> | ||
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- | <font size='3'>'''Abstract'''</font> | ||
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- | ==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 <ref name='genetics'>Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.</ref>. 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)<ref>2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial</ref>. 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. | ||
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- | Ribonucleotides enter through the secondary channel (15 x 20 Å)<ref name='loading'>PMID: 17581591</ref>. 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<ref name='loading' />. | ||
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- | 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<ref name='loading' />. 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 <ref name='loading' />. 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 <ref name='loading' />. | ||
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==References== | ==References== | ||
<references /> | <references /> |
Revision as of 03:22, 10 September 2013
Contents |
Alpha Subunit of Thermus aquaticus DNA Polymerase III
Introduction
Role of DNA Polymerase in Replication
The primary function of DNA polymerase is to replicate the DNA of an organism. DNA replication occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see DNA Replication, Transcription and Translation). DNA consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.
The replisome is an enzymatic complex with multiple subunits (see Replisome Diagram). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition and elongation of the growing DNA strand.
Prokaryotic DNA Polymerases
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication[1]. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated [1]. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear[1]. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far[1].
Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs [1]. DNA polymerase III is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of Thermus aquaticus, commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)[2]. Taq DNA polymerase III, a replicative polymerase, is homologous to that of Escherichia coli and also Polβ, a eukaryotic polymerase specializing in repair instead of replication [2].
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 of DNA Polymerase Functional Core
Video of DNA Polymerase Active Site
Important Domains
|
The fingers, palm and thumb domains of DNA polymerase III are highly conserved [3]. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. These domains form binding pockets for both DNA and incoming dNTPs[2]. They are also involved in correct dNTP selection, position and addition along with DNA interactions [2].
- Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved catalytic aspartates[2]. The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates)[2].
- Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group[2]. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis[2]. The fingers domain forms part of the active site[2].
- Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA[2].
- β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA[2].
- C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove[2].
- Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel[2]. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined[2].
- Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain[2].
DNA Interactions & Conformational Changes
The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove[2]. 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[2]. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA[2]. The β-binding domain, fingers and thumb also adjust[2].
Nucleotide Addition
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2012 UW-Milwaukee CREST Team
Team Members
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker
Abstract
ABSTRACT
References
- ↑ 1.0 1.1 1.2 1.3 1.4 Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm.
- ↑ 2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18 Wing RA, Bailey S, Steitz TA. Insights into the replisome from the structure of a ternary complex of the DNA polymerase III alpha-subunit. J Mol Biol. 2008 Oct 17;382(4):859-69. Epub 2008 Jul 27. PMID:18691598 doi:10.1016/j.jmb.2008.07.058
- ↑ Kohlstaedt LA, Wang J, Friedman JM, Rice PA, Steitz TA. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783-90. PMID:1377403 doi:[http://dx.doi.org/10.1126/science.1377403 http://dx.doi.org/10.1126/science.1377403