User:Luis E Ramirez-Tapia/T7 RNA polymerase
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[http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood. | [http://en.wikipedia.org/wiki/Transcription_(genetics) Transcription] is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[http://en.wikipedia.org/wiki/RNA_polymerase]''RNA polymerases''). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the [http://ecoliwiki.net/colipedia/index.php/Phage_T7 bacteriophage T7], is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the <scene name='User:Luis_E_Ramirez-Tapia/Sandbox_3/Initiation/2'>INITIATION</scene> phase (Figure 1) remains poorly understood. | ||
<br> | <br> | ||
| - | [[Image:Abortivecycling.png|thumb| | + | [[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/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> | ||
Revision as of 04:12, 29 April 2011
One of the CBI Molecules being studied in the University of Massachusetts Amherst Chemistry-Biology Interface Program at UMass Amherst and on display at the Molecular Playground
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Color code N-Terminus domain, Subdomain H, Helices C1 and C2, specificity loop, Non-template strand, template strand and the nascent RNA strand |
Contents |
Conformational Changes on T7 RNA Polymerase
Transcription is a fundamental part of genetic regulation. The RNA polymerases that accomplish this function vary in structure, size and complexity, but must all carry out the same basic functions ([See[1]RNA polymerases). The correct transcription of DNA to RNA depends of several factors and the complexity increases with the complexity of the organism. This makes the study of the transcriptional process complicated. The RNA polymerase of the bacteriophage T7, is the perfect model for studying the transcription process given that T7 RNA polymerase is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Nevertheless, there is plenty to learn from the transcription mechanism, such as the "abortive cycle" process that takes place during the phase (Figure 1) remains poorly understood.
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 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 . Another interesting observation that could help to resolve the mechanism of abortive cycling, is a single point mutation at the (notice the position of the ). This mutation is far away from the and the 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.
Understanding the morph
You can see the transition between the conformation and the complex by pressing the follow button. The first striking observation is the conformational change of the N-terminus part of the enzyme and the helices C1-C2. The DNA with translucent colors is our reference point, the modeled DNA is part of the intermediate state structure. The N-terminus rotates around 47º, the RNA transcript has 7 bases, still the enzyme has not reached its elongation conformation. The missing steps could be resolved if we morph the structures using the intermediate state structure and the elongation structure. The follow , shows a complete refolding of the sub-domain H (alfa-helices in green) and the helices C-1 C-2, it uses the intermediate state and the elongation state. However there is a problem. Could you see it? follow the green helices and you will see it. Yes, it can not a the real transition. Although there has been good advances in solving the correct transition (2), the optimal way, is by producing structures of the transitional complexes from 9 and 10 mer transcripts. Another approach will require the label of the enzyme with fluorophores, then using FRET we could calculate the distances and make a model of the correct transition. That is work in progress... Finally the morphs were produced using the energy minimization morphing software from the 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).
References
- Steitz, T. A. (2009) The structural changes of T7 RNA polymerase from transcription initiation to elongation., Curr. Opin. Struct. Biol. 19, 683-690.
- Turingan, R. S., Theis, K., and Martin, C. T. (2007) Twisted or shifted? Fluorescence measurements of late intermediates in transcription initiation by T7 RNA polymerase., Biochemistry 46, 6165-6168.
Acknowledgement
To Professor Eric Martz his advice was crucial to develop this page.
