This web page was produced as an assignment for an undergraduate course at Davidson College.

A Comparison of T7 RNA Polymerase Initiation and Elongation Phases



This is a tutorial for two conformations of T7 RNA polymerase. As you scroll down, you can read information about the molecule, as well as click on buttons which will orient the two molecules and highlight their attributes in informative ways. In both windows, the protein is depicted as blue, the DNA helix is depicted as light blue (template strand) and green (non-template strand), and the growing RNA strand is yellow.
These two boxes reset the images.


Click the next two buttons to see brief animations for each conformation, highlighting the topography of the molecules. Upon completion of the animation, the image will reset itself.


T7 RNA polymerase binds to specific DNA promoters in order to transcribe RNA, and in doing so, undergoes a transition from an initiation phase to an elongation phase, two seperate conformations which are quite different from one another, structurally and functionally. It is thought that the extreme change in conformation contributes to the processivity of the polymerase in its elongation phase. The thumb domains (residues 371-410) have been rotated to approximately the same orientation to highlight the conformation change. You can see that the thumb domain of the elongation complex is slightly more bent than the initiation complex. Click on both boxes to highlight the alignment of the thumb domains (in red ribbons).


Residues 230-240 constitute a structure called the intercalating hairpin. In the initiation complex, the intercalating hairpin opens the promoter. In the elongation phase, it becomes disordered and is removed from the vicinity of the DNA helix. This is apparent even from afar. Click these boxes to highlight the position of the intercalating hairpin (orange) for both conformations.


Click here to highlight the interaction between the intercalating hairpin and the DNA helix during initiation. The protein's display has been switched to space-filling mode to emphasize the intercalating hairpin's position between the strands of the duplex.


Click here to zoom out again:


The specificity loop (residues 740-770) is a structure which makes sequence-specific interactions with the DNA's promoter in the initiation phase. In the elongation complex, the specificity loop's position shifts, allowing a tunnel to form through which RNA exits. Click the next two boxes to highlight the location of the specificity loop in magenta.


In the initiation complex, the specificity loop's interactions with DNA occur in its major groove. Click the left box to zoom in on these interactions. Click the right box to zoom in on the specifity loop's role as part of the exit tunnel in the elongation complex.


Click the next two boxes to zoom out again.


One of the other major conformational changes involves a structure called helix C (residues 31-61). In the initiation complex, helix C is bent into an L-shape. In the elongation complex, the two portions are stacked on top of each other to form one long helix. Click the next two boxes to see the difference between conformations. Helix C will be displayed as white ribbons.


Another structure which undergoes a significant conformational change is Subdomain H (residues 160-190). In the initiation complex subdomain H is on the outskirts of the protein and does not have an active role. However, it is refolded in the elongation complex into a pair of anti-parallel helices which form the inner portion of the RNA exit tunnel. Click the buttons to highlight Subdomain H in violet. In the lefthand panel, it is located in the bottom-left corner. In the righthand panel, it can be seen in the upper-right.


Clicking the next two buttons hides the protein and displays only the nucleic acids. In the left panel, you can see that the duplex has been completely seperated and that two molecules of GTP are getting ready to be added onto the template strand. In the right hand column, you can see that the 3' end of the RNA strand is bound to the template strand and that the 5' end of the RNA strand is twisting off the helix. Also clearly depicted in the right frame is the "transcription bubble." This is a portion of denatured DNA, flanked by bound DNA, where transcription occurs.


The next two boxes zoom in on the interaction between the DNA template strand and the RNA transcript. In the left panel, you can see two molecules of GTP that have not yet been hydrogen bound to the template strand in the initiation phase of T7 RNAP. In the right panel, you can see that hydrogen bonds have formed between the 3' end of the transcipt and the template strand of DNA.


Click the next two buttons to orient the protein to the perspective of the DNA helix. You can see the intercalating hairpin (still orange) dividing the two strands of DNA in the initiation complex. In the elongation complex, you can see the double-stranded DNA entering the protein at the left, and exiting at the right.


Click the next button to rotate the protein and home in on the RNA exit tunnel, which is on the reverse side.


This concludes the tutorial. Visit the Protein Data Bank

Reference:

Yin, Whitney Y. and Thomas A. Steitz. "Structural Basis for the Transition from Initiation to Elongation in T7 RNA Polymerase." Science. Vol 298, pages 1387-1394.

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