User:Luis E Ramirez-Tapia/Sandbox 3
From Proteopedia
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 |
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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. The correct transcription of DNA to RNA depends of several transcription factors and the complexity increases with the complexity of the organism, making the study of the transcriptional process more complicated. The RNA polymerase of the bactereophage T7, is the perfect model for studying the transcription process. The main reason, it is a single unit enzyme that processes RNA with the same effectivity as the polymerase from higher organisms. Still much of the mechanism is unknown, for example, during the step, there is an event called "abortive cycling" (Figure 1.). In this event the small RNA transcripts (less than 12 bases) fall from the complex. The abortive cycling will continue until the enzyme/DNA/RNA complex reach the phase, where a more stable enzyme/DNA/RNA complex is form. A mayor contributor of the stability of the complex is the formation of the . Other interesting point is a single mutation at the that has been reported to decrease the amount of abortive cycling. We are focus in resolved the mechanism behind this mutation.
Understanding the morph
This morph was produced using a energy minimization morphing software (Yale Morph), the structures were the INITIATION (PDB ID: 1qln) and the INTERMIDATE STATE (PDB ID: 3e2e) complexes. One of the most striking characteristics of this enzyme is the HUGE conformational change of the N-terminus part of the enzyme, the Subdomain H (alfa-helices in green) and the helices C1-C2. Recently a new structure from an intermediate state (PDB ID:3e2e) was resolved (1). You can see the transition between the conformation and the complex by pressing the follow button. The DNA with translucent color is our reference point and it is part of the intermediate state structure. The N-terminus rotates around 47ยบ, the RNA transcript has 7 bases, still the enzyme has not reach it's elongation conformation. The missing details could be resolved if we try to morph the structures using the intermediate state structure and the elongation structure. I produced this , using the transition state and the elongation state, however an interesting event happen. Could you see what is the problem?. This is your homework. Although there has been good advances in solving how the correct transition (2), the correct transition could be resolved if others intermediate states from transcripts of 9 and 10 mer are resolved. Other approach requires 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.
