Posted by: Dan | July 23, 2006

An Insight into Ribozyme Catalysis

Via Ivy Privy, I was drawn back to a paper in the most recent issue of Cell that I had skipped over – Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis, explained more simply by Science Daily.

While the Cell paper may be a tough read for those not familiar with structural biology, and it doesn’t finalize the question of how life began, it does give some insights into the “RNA World” hypothesis: Martick and Scott used time-resolved crystallography to reconcile mechanistic explanations for ribozyme catalysis that, until recently, held serious discrepancies.

The solution, they found, were additional contacts at the active site that were acquired by previously minimal ribozymes, resulting in a 1000-fold catalytic enhancement. Further, Martick and Scott advance the case for simple acid-base chemistry as the basis for organic chemistry at the dawn of life.

Image and link, courtesy of William Scott, below the fold:

acid-base
(click on the image for more)


Responses

  1. A quibble: I don’t think this study was based on time-resolved crystallography. The introduction just mentions previous work done using that technique.

  2. Right… sorry about that. It does specify “macromolecular” crystallography, with a resolution of 2.2A, which I assume is different from time-resolved.

    I’m not up on the details of the various crystallography techniques out there, hence my confusion… you wouldn’t by chance be able to explain it better for me, would you?

  3. “Macromolecular” just means big molecules. The field was frequently called “protein crystallography” until people started doing structures with RNA and DNA in them.

    Their structure was static. Their molecule was not changing during the course of the experiment. I haven’t read the paper in detail, nor the previous studies to which they refer, so I don’t know precisely what they saw in their structure that wasn’t seen before. In looking over the “Experimental Procedures” section, it says the crystals took 12 months to grow! There is no specific mention of what temperature the X-ray diffraction data was collected at (100 K is typical), but it does mention “cryofreezing”, so 100 K is a reasonable guess.

    2.2 A resolution should be sufficient to locate most of the non-hydrogen atoms in the structure. It is still possible that surface loops or entire sections of a molecule may not be ordered in the crystal lattice, and thus do not show up in the X-ray structure. Maybe this is what happened in previous attempts at hammerhead structures. Either the 12 months of crystal growth gave the molecule time to order itself, or else some slow RNA lysis changed the constituency of the RNA to allow something to rearrange.

    In time-resolved crystallography, an attempt is made to capture transient states or reaction intermediated by syncing a trigger event, typically a laser to photolyse some sensitive substrate, with fast X-ray data collection. A wider range of X-ray energies is used with Laue diffraction to get as much data as quickly as possible. The need for reproducible triggering limits the number of candidate systems for time-resolved crystallography.

  4. W. Scott, PI for the study that this post is about, has himself entered a comment under the Link Digest post where Ivy Privy tipped me off to the article:

    Thanks for posting this. It is almost as much an honor to be featured on an atheist website as it is to get death threats from creationists.

    The link http://xanana.ucsc.edu/hh/full_length_hammerhead.html gets you to some more information and molecular structures that you can actually play around with in your web browser (assuming it has javascript). The paper itself by the way is freely available on http://www.cell.com. I am not sure if that will persist, but if not I will make a pdf available to anyone from our website.

  5. Again, thanks for the write-up. Our hope is in fact to do some time-resolved crystallography on this system, which will likely be a lot more interesting than our previous attempts.

    Basically the current structure is like one instantaneous snapshot at the beginning of the reaction, one that is three-dimensional, and high enough in resolution that you can see where all the atoms are. Our hope is that we might be able to get a series of snapshots at various steps in the reaction.

    A totally separate project that is currently underway in our lab involves trying to get a series of snapshots of structures during an in vitro evolution process in which a ribozyme becomes a better catalyst.

  6. 2.2 A resolution should be sufficient to locate most of the non-hydrogen atoms in the structure. It is still possible that surface loops or entire sections of a molecule may not be ordered in the crystal lattice, and thus do not show up in the X-ray structure. Maybe this is what happened in previous attempts at hammerhead structures. Either the 12 months of crystal growth gave the molecule time to order itself, or else some slow RNA lysis changed the constituency of the RNA to allow something to rearrange.

    Just to clarify: All the atoms are well-ordered and are clearly visible.

    What happened previously is that we went after a piece of the RNA that was too small. It turns out we, along with everyone else in the field, ignored a more distant tertiary contact in the RNA that stabilizes the active site.

    The best way to see the differences is with this movie (quicktime), which represents a morphing of the older structure into the newer one.

    http://xanana.ucsc.edu/hh/figs/supplemental_materials/morphing_structures.mov


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