|
|
| Line 3: |
Line 3: |
| | <!-- INSERT YOUR SCENES AND TEXT BELOW THIS LINE --> | | <!-- INSERT YOUR SCENES AND TEXT BELOW THIS LINE --> |
| | | | |
| - | =='''EcoRV endonuclease'''== | |
| | | | |
| - | <Structure load='1rva' size='500' frame='true' align='right' caption='EcoRV is a restriction enzyme found in escherichia coli bacteria. It can be thought of as scissors for cutting DNA and applications are being researched in gene analysis and cloning.' scene='Insert optional scene name here' />
| + | =='''YourMacromolecule'''== |
| | | | |
| | ===Introduction=== | | ===Introduction=== |
| - | | + | <Structure load='1a84' size='300' frame='true' align='right' caption='pdbcode, Insert caption here' scene='Insert optional scene name here' /> |
| - | EcoRV endonuclease is a type II restriction enzyme, or restriction endonuclease, found in e. coli bacteria. The main biological function of restriction endonucleases is to protect the cell's genome against any foreign DNA. Restriction enzymes recognize and cleave specific sequences of DNA. In the figure to the right, the enzyme is shown with the <scene name='Sandbox_Reserved_428/Dna_sequence/1'>eleven base sequence</scene> AAA<font color=red>GATATC</font>TT, which includes the recognition site <font color=red>GATATC</font>. The recognition site is shown <scene name='Sandbox_Reserved_428/Gatatc/1'>here</scene>. A type II restriction enzymes cleave at a short distance from the recognition site and often use Mg(2+) as a cofactor, as does this enzyme. They are commonly found in bacteria and shared structural features indicate that they are evolutionarily related.<ref>PMID:7819264</ref> Type II endonucleases have been the site of much research because of applications in gene analysis and cloning and because they are great at modeling protein-DNA interactions.<ref>PMID:11557805</ref>
| + | <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> |
| - | | + | |
| - | Specifically, EcoRV is an orthodox restriction endonuclease. This means that the DNA sequence recognized is palindromic, meaning each strand contains the same sequence.<ref>PMID:11557805</ref> The DNA duplex is cleaved at the phosphodiester bond located at 5'-<font color=red>GAT</font><font color=blue>*</font><font color=red>ATC</font>-3'. The other DNA strand will also be cleaved in the same location, producing blunt ends. The cleavage site is shown <scene name='Sandbox_Reserved_428/Cleavage_site/1'>here</scene>. This is relatively unique among restriction enzymes, as many cleave each DNA stand at a different location, leaving what are known as sticky ends. Cleavage occurs by the breaking of the bond between a 3' oxygen and the phosphorus by nucleophilic attack by water.<ref>PMID:11557805</ref> Mg(2+) acts as a catalyst for this reaction, however it is not shown in the representation to the right.<ref>PMID:7819264</ref> The process of DNA cleavage is covered in more detail in the Binding Interactions section.
| + | |
| - | | + | |
| - | The steps of DNA cleavage are as follows. EcoRV binds to the DNA without specificity, which is followed by a diffusional walking "search" down the DNA molecule. If the protein encounters its recognition site, conformational changes occur in the enzyme-DNA complex.<ref>PMID:11557805</ref> These changes are discussed in more depth in the additional features section.
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | .
| + | |
| | | | |
| | ===Overall Structure=== | | ===Overall Structure=== |
| - | <Structure load='1rva' size='500' frame='true' align='right' caption='EcoRV' scene='Insert optional scene name here' /> | + | <Structure load='1a84' size='300' frame='true' align='right' caption='pdbcode, insert caption here' scene='Sandbox_Reserved_430/Intra-strand_phosphate/1' /> |
| - | EcoRV endonuclease is functional as a <scene name='Sandbox_Reserved_428/Dimer_complex/4'>dimer</scene> consisting of two monomers; <font color='limegreen'>monomer B</font> and <font color='violet'>monomer A</font> are in a U shape. Each monomer consists of 245 amino acids arranged in alpha/beta secondary structures. These monomers are identical in their sequencing but not their structure.
| + | <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> |
| - | | + | |
| - | The monomer's structure are identical in all but two sets of residue numbers. There are 9 identical <font color='crimson'>alpha helices</font> shown <scene name='Sandbox_Reserved_428/Dimeralphahelix/2'>here</scene> and 10 identical <font color='aqua'>beta strands</font> shown <scene name='Sandbox_Reserved_428/Dimerbetasheets/2'>here</scene>.
| + | |
| - | | + | |
| - | The structures <scene name='Sandbox_Reserved_428/Diff_dimer/2'>differ</scene> at residue number 144-150, where <font color='violet'>monomer A</font> has an additional <font color='dark orange'>alpha helix</font> and a residue numbers 150-153, where <font color='limegreen'>monomer B</font> has an additional <font color='navy'>beta strand</font>.
| + | |
| - | | + | |
| - | At the point on the U dimer where the monomers meet there is a 5 strand <scene name='Sandbox_Reserved_428/Dimer_connection_betasheet/1'>interlocking beta sheet</scene>. This anti-parallel beta sheet is made of 3 stands from <font color='limegreen'>monomer B</font> and 2 strands from <font color='violet'>monomer A</font>. This beta sheet also has three alpha helices packed against it shown <scene name='Sandbox_Reserved_428/Alpha_beta_sandwhich/2'>here</scene>. Two of these alpha helicies are from <font color='violet'>monomer A</font> and the other is from <font color='limegreen'>monomer B</font> This structure assists in the structure and stability of the dimer as a whole. Once you <scene name='Sandbox_Reserved_428/Zoomout_alpha_beta_sandwhich/1'>zoom out</scene>, you can see how these beta strands form one anti-parallel beta sheet connecting the two monomers at the bottom of the U shape.
| + | |
| - | | + | |
| - | There are two structural sub-domains. The first, called the <font color='orange'>dimerization sub-domain</font>,shown <scene name='Sandbox_Reserved_428/Sub-domain_dimer/4'>here</scene>, is the smallest of the sub-domains, residue numbers 19-32, 150-160, and 144-150 of <font color=violet>monomer A</font>. This section forms all of the dimer interface interactions and stabilizes the dimer. This sub-domain is unique to EcoRV and only one other restriction endonuclease. The second is called the <font color='deep pink'>DNA binding sub-domain</font> shown <scene name='Sandbox_Reserved_428/Sub-domain_dimer/2'>here</scene>, residue numbers 2-18 38-140 and 167-243. This sub-domain is where the majority of the dimer's function occurs. This sub-domain contains the loops that are responsible for the interactions with DNA which include binding to and breaking it's bonds. The remaining segments of amino acids make up the <font color='mediumspringbreeze'>flexible linkage</font> between the two sub-domains, residue numbers 33-37,141-143, and 161-165 shown <scene name='Sandbox_Reserved_428/Sub-domain_dimer/5'>here</scene>. This flexibility allows for the enzyme to open up and the monomers to slightly separate during the free enzyme form.
| + | |
| - | <ref>PMID: 8491171</ref>
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | .
| + | |
| | | | |
| | ===Binding Interactions=== | | ===Binding Interactions=== |
| - | <Structure load='1rva' size='500' frame='true' align='right' caption='EcoRV' scene='Insert optional scene name here' /> | + | <Structure load='1a84' size='300' frame='true' align='right' caption='pdbcode, Insert caption here' scene='Insert optional scene name here' /> |
| - | | + | <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> |
| - | | + | |
| - | The EcoRV molecule is a type II restriction endonuclease, which means that its purpose is to cleave DNA at the center of its <scene name='Sandbox_Reserved_428/Recognition_sequence/3'>recognition sequence</scene>, which is the site that the enzyme recognizes on the DNA to be cleaved. DNA recognition sites on the EcoRV molecule, called R-loops, bind to the major grooves of the double stranded DNA at its recognition sequence, which is <font color='yellow'>GATATC,</font> by hydrogen bonding. These hydrogen bonds make the DNA form a kinked conformation that is later stabilized by the addition of the Mg2+ ion. The Mg2+ ion is a catalyst that causes the DNA to shift in a way that increases the rate necessary for the DNA cleavage. It does not bind to EcoRV until the enzyme is prepared to cleave the DNA helix. When the enzyme is ready to cleave, the affinity for the Mg2+ ion increases and then it binds to the enzyme. The recognition sequence of the DNA is shown in yellow and it is cleaved at the center <font color='darkmagenta'>between the T and A base pairs</font> on the D chain when interacted with a Mg2+ ion, leaving a blunt end in the DNA.
| + | |
| - | | + | |
| - | The <scene name='Sandbox_Reserved_428/Active_site/3'>active site</scene> of the enzyme is shown here in pink. It consists of four residues, <font color='fuchsia'> Asp74, Asp90, Ile91, and Lys92, </font> which participate in the binding of the Mg2+ ion along with binding interactions of the Adenine, A, base pair. Mg2+ binding only occurs in <scene name='Sandbox_Reserved_428/Active_site_one_subunit/1'>subunit B</scene> along one side of the DNA double helix. The Mg2+ binding site is formed when ionic interactions cause the slightly acidic Asp90 residue and the slightly negatively charged scissile phosphodiester group on the DNA strand to approach each other. This allows the Mg2+ ion to bind to this enzyme, also with ionic interactions between the positively charged Mg2+ and the partially negative charged <scene name='Sandbox_Reserved_428/6_oxygens_that_bind_to_mg2/2'>oxygen atoms</scene>. These molecules that bind to the Mg2+ ion are the <font color='red'>carboxylate oxygen atoms</font> from the Asp74 and Asp90 residues, the <font color='red'>nonesterified oxygen</font> from the scissile phosphodiester group, and three additional <font color='blue'>oxygen atoms from three water molecules</font>. These six ionic bonds form an octahedral shape in the active site of this enzyme.
| + | |
| - | | + | |
| - | These six ionic interactions all have about the same binding distance except for one bond between the oxygen from the <scene name='Sandbox_Reserved_428/Asp74_molecule_farther/1'>Asp74 residue</scene> and the Mg2+ ion that is significantly longer. The five similar bond lengths are all about 2.08 Å, but the bond between Mg2+ and the Asp74 oxygen spans a distance of 2.9 Å. This is noted because the Asp90 and scissile phosphodiester molecules that bind to this Mg2+ ion change their bonding interactions with hydrogen to accommodate the addition of the Mg2+ ion. The Asp74 residue maintains its hydrogen bond interactions on its side chain with the main chain of the Ile91 residue and the water molecule, which is why it keeps a greater distance between itself and the Mg2+ ion.<ref>PMID:7819264</ref>
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | .
| + | |
| | | | |
| | ===Additional Features=== | | ===Additional Features=== |
| - | <Structure load='1rva' size='500' frame='true' align='right' caption='EcoRV' scene='Insert optional scene name here' /> | + | <Structure load='1a84' size='300' frame='true' align='right' caption='pdbcode, Insert caption here' scene='Insert optional scene name here' /> |
| | + | <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> |
| | | | |
| - | As a prerequisite for efficient target site recognition, the enzyme is capable of nonspecific binding with a DNA molecule's Phosphate backbone. The non-specific Enzyme(PDB: 2RVE) slides along a DNA molecule with movement characterized by a helical diffusion due to hydrogen bonding along the minor groove . The biological significance of this process is accelerated target site location, increased processivity, and dissociation of the enzyme upon cleavage. Specific binding on the other hand is precisely the process of recognition which involves an interplay between interaction with the bases of the recognition sequence as well as indirect interaction with the respective phosphate backbone. Recognition precedes conformational changes in both DNA and the protein, thereby bending the DNA and activating the catalytic centers. In both the <scene name='Sandbox_Reserved_428/Specific_minorgroove/2'>specific</scene> and <scene name='Sandbox_Reserved_428/Ns_minorgroove_trackng/2'>Non-Specific</scene> complexes, the DNA is oriented so that the minor groove faces the <font color='blue'> floor</font> of the Binding site.<ref>PMID:11557805</ref>
| + | ===Quiz Question 1=== |
| | + | <Structure load='1a84' size='300' frame='true' align='right' caption='pdbcode, Insert caption here' scene='Insert optional scene name here' /> |
| | + | <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> |
| | | | |
| - | Unlike <font color ='red'>R-loops</font> (Recognition Loops, Residues 182-187) which are disordered in both the <scene name='Sandbox_Reserved_428/Nonspecificenzyme_qloops/1'>Non-Specific</scene> and <scene name='Sandbox_Reserved_428/Ns_rloops/1'>free enzyme</scene> complex(PDB: 1RVE). <font color='yellow'>Q-Loops</font> (Residues 68-71) are ordered in the <scene name='Sandbox_Reserved_428/Ns_qloops/1'>non-specific complex</scene>, forming a beta turn. They are disordered in the <scene name='Sandbox_Reserved_428/Qloops_freeenzyme/1'>free enzyme</scene>. Hydrogen bond interactions in the non-specific complex are indicated in crystal structures of the <scene name='Sandbox_Reserved_428/Nonspecific_gah/1'>non-specific complex</scene> between the two <font color='Blue'>Glutamine</font>, <font color='red'>Aspargarine</font>, and <font color='green'>Histidine</font> Residues of the Q-Loops and the DNA Phosphates. While <font color='green'>Histidine</font> and the two <font color='Blue'>Glutamine</font> residues contribute less to hydrogen bonding due to their high conformational entropy, <font color='red'>Aspargarine</font> forms the strongest hydrogen bonds with the Phosphates because of its short alkyl chain, and thus is a strong contributor to the ability of the Enzyme to diffuse Linearly along the DNA molecule.<ref>PMID: 8491171</ref>
| + | ===Quiz Question 2=== |
| | + | <Structure load='1a84' size='300' frame='true' align='right' caption='pdbcode, Insert caption here' scene='Insert optional scene name here' /> |
| | + | <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> |
| | | | |
| | + | ===Credits=== |
| | | | |
| | + | Introduction - name of team member |
| | | | |
| | + | Overall Structure - name of team member |
| | | | |
| | + | Drug Binding Site - name of team member |
| | | | |
| | + | Additional Features - name of team member |
| | | | |
| | + | Quiz Question 1 - name of team member |
| | | | |
| | + | Quiz Question 2 - name of team member |
| | | | |
| - | | + | ===References=== |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | .
| + | |
| - | | + | |
| - | =='''References'''== | + | |
| | <references/> | | <references/> |
| - | | |
| - | =='''Credits'''== | |
| - | | |
| - | Introduction - Jesse Guillet | |
| - | | |
| - | Overall Structure - Nicole Bundy | |
| - | | |
| - | Binding Interactions - Julia Tomaszewski | |
| - | | |
| - | Additional Features - Sam Kmail | |
