User:Luis E Ramirez-Tapia/T7 RNA polymerase - Revision history

Luis E Ramirez-Tapia at 05:21, 29 April 2011

2011-04-29T05:21:22Z

←Older revision		Revision as of 05:21, 29 April 2011
Line 7:		Line 7:
	Color code:		Color code:
	<br>		<br>
-	<font color='magenta'><b>N-Terminus ~~domain~~</b></font>,	+	<font color='magenta'><b>N-Terminus Domain</b></font>,
	<font color='green'><b>Subdomain H</b></font>,		<font color='green'><b>Subdomain H</b></font>,
	<font color='orange'><b>Helices C1 and C2</b></font>,		<font color='orange'><b>Helices C1 and C2</b></font>,
-	<font color='yellow'><b>~~specificity loop~~</b></font>,	+	<font color='yellow'><b>Specificity Loop</b></font>,
-	<font color='x00ff00'><b>Non-template ~~strand~~</b></font>,	+	<font color='x00ff00'><b>Non-template Strand</b></font>,
-	<font color='x6060ff'><b>~~template strand~~</b></font> and the	+	<font color='x6060ff'><b>Template Strand</b></font> and the
-	<font color='red'><b>~~nascent~~ RNA ~~strand~~</b></font>	+	<font color='red'><b>Nascent RNA Strand</b></font>
	*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>		*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/3merrna/1'>Initiation state = 3 mer RNA</scene>
	*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>		*<scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/7merrna/1'>Intermediate state = 7mer RNA</scene>

Luis E Ramirez-Tapia at 05:20, 29 April 2011

2011-04-29T05:20:15Z

←Older revision		Revision as of 05:20, 29 April 2011
Line 5:		Line 5:
	</td></tr>		</td></tr>
	<tr><td>		<tr><td>
-	Color code	+	Color code:
		+	<br>
	<font color='magenta'><b>N-Terminus domain</b></font>,		<font color='magenta'><b>N-Terminus domain</b></font>,
	<font color='green'><b>Subdomain H</b></font>,		<font color='green'><b>Subdomain H</b></font>,

Luis E Ramirez-Tapia: /* Understanding the Morph */

2011-04-29T05:19:02Z

Understanding the Morph

←Older revision		Revision as of 05:19, 29 April 2011
Line 39:		Line 39:
	<p>The first striking observation is the conformational change of the <font color='magenta'>N-terminus</font> part of the enzyme and the <font color='orange'>helices C1-C2</font>.		<p>The first striking observation is the conformational change of the <font color='magenta'>N-terminus</font> part of the enzyme and the <font color='orange'>helices C1-C2</font>.
	The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The <font color='magenta'>N-terminus</font> rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the <font color =green> sub-domain H</font> (alfa-helices in green) and the <font color = orange>helices C-1 C-2</font>. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it?<b> Follow the movement of the green helices</b>. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...		The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The <font color='magenta'>N-terminus</font> rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the <font color =green> sub-domain H</font> (alfa-helices in green) and the <font color = orange>helices C-1 C-2</font>. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it?<b> Follow the movement of the green helices</b>. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...
-	Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were used in the server are the following: initiation ~~state~~ (PDB ID: 1qln), intermediate state (PDB ID: 3e2e) (1) and the elongation ~~state~~ (PDB ID:1msw).</p>	+	Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were used in the server are the following: T7 RNA polymerase initiation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1QLN (PDB ID: 1qln)], T7 intermediate state complex [http://www.pdb.org/pdb/explore/explore.do?structureId=3E2E (PDB ID: 3e2e)](1) and the T7 RNA polymerase elongation complex [http://www.pdb.org/pdb/explore/explore.do?structureId=1MSW (PDB ID:1msw)].</p>

	=References=		=References=

Luis E Ramirez-Tapia: /* Understanding the Morph */

2011-04-29T05:14:55Z

Understanding the Morph

←Older revision		Revision as of 05:14, 29 April 2011
Line 39:		Line 39:
	<p>The first striking observation is the conformational change of the <font color='magenta'>N-terminus</font> part of the enzyme and the <font color='orange'>helices C1-C2</font>.		<p>The first striking observation is the conformational change of the <font color='magenta'>N-terminus</font> part of the enzyme and the <font color='orange'>helices C1-C2</font>.
	The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The <font color='magenta'>N-terminus</font> rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the <font color =green> sub-domain H</font> (alfa-helices in green) and the <font color = orange>helices C-1 C-2</font>. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it?<b> Follow the movement of the green helices</b>. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...		The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The <font color='magenta'>N-terminus</font> rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the <font color =green> sub-domain H</font> (alfa-helices in green) and the <font color = orange>helices C-1 C-2</font>. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it?<b> Follow the movement of the green helices</b>. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...
-	Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were ~~feeded~~ are. initiation state (PDB ID: 1qln), intermediate state (PDB ID: 3e2e) (1) and the elongation state (PDB ID:1msw).</p>	+	Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were used in the server are the following: initiation state (PDB ID: 1qln), intermediate state (PDB ID: 3e2e) (1) and the elongation state (PDB ID:1msw).</p>

	=References=		=References=

Luis E Ramirez-Tapia: /* Understanding the Morph */

2011-04-29T05:11:55Z

Understanding the Morph

←Older revision		Revision as of 05:11, 29 April 2011
Line 38:		Line 38:
	<br>		<br>
	<p>The first striking observation is the conformational change of the <font color='magenta'>N-terminus</font> part of the enzyme and the <font color='orange'>helices C1-C2</font>.		<p>The first striking observation is the conformational change of the <font color='magenta'>N-terminus</font> part of the enzyme and the <font color='orange'>helices C1-C2</font>.
-	The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The <font color='magenta'>N-terminus</font> rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the <font color =green> sub-domain H</font> (alfa-helices in green) and the <font color = orange>helices C-1 C-2</font>. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it?<b> Follow the movement of the green helices</b>. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us calculate the distances ~~occurring~~ during the transition. This work in progress...	+	The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The <font color='magenta'>N-terminus</font> rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the <font color =green> sub-domain H</font> (alfa-helices in green) and the <font color = orange>helices C-1 C-2</font>. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it?<b> Follow the movement of the green helices</b>. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be by labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us to calculate the movement distances that occurs during the transition. This work is in progress...
-	Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]~~, the~~ structures that were ~~used~~ are ~~the INITIATION STATE~~ (PDB ID: 1qln), ~~the INTERMIDATE STATE~~ (PDB ID: 3e2e) (1) and the ~~ELONGATION STATE~~ (PDB ID:1msw).</p>	+	Finally, the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server]. The protein structures that were feeded are. initiation state (PDB ID: 1qln), intermediate state (PDB ID: 3e2e) (1) and the elongation state (PDB ID:1msw).</p>

	=References=		=References=

Luis E Ramirez-Tapia: /* Understanding the Morph */

2011-04-29T05:04:25Z

Understanding the Morph

←Older revision		Revision as of 05:04, 29 April 2011
Line 27:		Line 27:
	----		----
	===Understanding the Morph===		===Understanding the Morph===
-	In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, ~~press~~ the following button.	+	In order to activate the transition between the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> conformation and the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Intermediate_state/4'>INTERMEDIATE STATE</scene> complex, click the following button.
	<jmol>		<jmol>
	<jmolButton>		<jmolButton>

Luis E Ramirez-Tapia: /* Conformational Changes on T7 RNA Polymerase */

2011-04-29T05:02:52Z

Conformational Changes on T7 RNA Polymerase

←Older revision		Revision as of 05:02, 29 April 2011
Line 24:		Line 24:
	[[Image:Abortivecycling.png\|thumb\|400px\|left\|<b> Figure 1. Abortive Cycle during transcription initiation</b>]]		[[Image:Abortivecycling.png\|thumb\|400px\|left\|<b> Figure 1. Abortive Cycle during transcription initiation</b>]]

-	<p>In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene> ~~(notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>)~~. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.</p>	+	<p>In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>. This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). Part of our research is focused on resolving the mechanism behind this mutation.</p>
	----		----
	===Understanding the Morph===		===Understanding the Morph===

Luis E Ramirez-Tapia: /* Conformational Changes on T7 RNA Polymerase */

2011-04-29T05:00:44Z

Conformational Changes on T7 RNA Polymerase

←Older revision		Revision as of 05:00, 29 April 2011
Line 24:		Line 24:
	[[Image:Abortivecycling.png\|thumb\|400px\|left\|<b> Figure 1. Abortive Cycle during transcription initiation</b>]]		[[Image:Abortivecycling.png\|thumb\|400px\|left\|<b> Figure 1. Abortive Cycle during transcription initiation</b>]]

-	<p>In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.</p>	+	<p>In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.</p>
	----		----
	===Understanding the Morph===		===Understanding the Morph===
Line 38:		Line 38:
	<br>		<br>
	<p>The first striking observation is the conformational change of the <font color='magenta'>N-terminus</font> part of the enzyme and the <font color='orange'>helices C1-C2</font>.		<p>The first striking observation is the conformational change of the <font color='magenta'>N-terminus</font> part of the enzyme and the <font color='orange'>helices C1-C2</font>.
-	The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The <font color='magenta'>N-terminus</font> rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the <font color =green> sub-domain H</font> (alfa-helices in green) and the <font color = orange>helices C-1 C-2</font>. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it?<b> Follow the movement of the green helices</b>. Indeed, it can not be a the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us calculate the distances occurring during the transition. This work in progress...	+	The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The <font color='magenta'>N-terminus</font> rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the <font color =green> sub-domain H</font> (alfa-helices in green) and the <font color = orange>helices C-1 C-2</font>. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it?<b> Follow the movement of the green helices</b>. Indeed, it can not be the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us calculate the distances occurring during the transition. This work in progress...
	Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).</p>		Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).</p>

Luis E Ramirez-Tapia: /* Conformational Changes on T7 RNA Polymerase */

2011-04-29T04:59:47Z

Conformational Changes on T7 RNA Polymerase

←Older revision		Revision as of 04:59, 29 April 2011
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	[[Image:Abortivecycling.png\|thumb\|400px\|left\|<b> Figure 1. Abortive Cycle during transcription initiation</b>]]		[[Image:Abortivecycling.png\|thumb\|400px\|left\|<b> Figure 1. Abortive Cycle during transcription initiation</b>]]

-	<p>In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/~~proline266~~/1'>proline 266</scene> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.</p>	+	<p>In this event the small RNA transcripts (less than 12 bases) dissociate from the complex. The abortive cycle will continue until the enzyme/DNA/RNA complex reaches the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/1mswcolor/2'>ELONGATION </scene> phase in order to for a more stable enzyme/DNA/RNA complex. A mayor contributor of the stability of the complex is the formation of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Exit_tunnel/3'>RNA exit tunnel</scene>. Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/P266l/1'>proline 266</scene>> (notice the position of the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Transition/2'>P266L mutation during the transition</scene>). This mutation is far away from the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Activesite/2'>active site</scene> and the <scene name='User:Luis_E_Ramirez-Tapia/T7_RNA_polymerase/Promotercontact/1'>promoter contact</scene> region and it is located on the hinge between the N-terminus and the C-terminus. Although leucine is not the only substitution that decreases the amount of abortive products, it is the one with the mayor effect. It is proposed that the mutation creates a more flexible protein structure that facilitates the transition from initiation to elongation. Part of our research is focused on resolving the mechanism behind this mutation.</p>
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	===Understanding the Morph===		===Understanding the Morph===

Luis E Ramirez-Tapia: /* Understanding the Morph */

2011-04-29T04:51:03Z

Understanding the Morph

←Older revision		Revision as of 04:51, 29 April 2011
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	<br>		<br>
	<p>The first striking observation is the conformational change of the <font color='magenta'>N-terminus</font> part of the enzyme and the <font color='orange'>helices C1-C2</font>.		<p>The first striking observation is the conformational change of the <font color='magenta'>N-terminus</font> part of the enzyme and the <font color='orange'>helices C1-C2</font>.
-	The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The <font color='magenta'>N-terminus</font> rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the <font color =green> sub-domain H</font> (alfa-helices in green) and the <font color = orange>helices C-1 C-2</font>. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it?<b> Follow the movement of the green helices</b>. Indeed, it can not be a the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach ~~could require the label of~~ the enzyme with fluorophores, then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET] we could calculate the distances ~~and make a model of~~ the ~~correct~~ transition. ~~That is~~ work in progress...	+	The DNA with translucent colors is our reference point and the modeled DNA is part of the intermediate state structure. The <font color='magenta'>N-terminus</font> rotates around 47º, the RNA transcript has 7 bases, but the enzyme has not reached its final elongation conformation yet. The missing steps could be resolved if we morph the structures using the intermediate state and the elongation structures. The following <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/T7wrongtransition/1'> most notorious conformational change</scene> shows a complete refolding of the <font color =green> sub-domain H</font> (alfa-helices in green) and the <font color = orange>helices C-1 C-2</font>. It uses the intermediate state and the elongation state. However, there is a problem. Can you see it?<b> Follow the movement of the green helices</b>. Indeed, it can not be a the real transition. While there has been good advances in solving the correct transition [http://www.ncbi.nlm.nih.gov/pubmed/17472344 (2)], the optimal way is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach to study this transition would be labeling the enzyme with fluorophores and then using [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer FRET], which could allow us calculate the distances occurring during the transition. This work in progress...
	Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).</p>		Finally the morphs were produced using the energy minimization morphing software from the [http://molmovdb.mbb.yale.edu/molmovdb/morph/ Yale Morph Server], the structures that were used are the INITIATION STATE (PDB ID: 1qln), the INTERMIDATE STATE (PDB ID: 3e2e) (1) and the ELONGATION STATE (PDB ID:1msw).</p>