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Self-capping RNA catalysts derived from selection-amplification.

The reactions of nucleoside 5[prime] triphosphates are among the most widespread (and therefore ancient) of the reactions of modern biochemistry. To study RNA enzymes that catalyze an important class of such reactions, we devised a new selection for RNAs that labilize their [Beta][Gamma] phosphates. We select RNAs that circularize in the presence of T4 RNA ligase, a reaction that requires a free 5[prime] [Alpha]-phosphate and therefore indicates release of the [Beta][Gamma] phosphates present in every transcript. Unexpectedly, free phosphates were found to react readily at the [Alpha]-phosphate of such RNAs. This was emphasized by further selection for RNAs that self-link to the free triphosphate of UTP-agarose. The most prevalent ribozyme (40% of the final, doubly-selected pool) has been characterized.

The uniform product of all such reactions is released pyrophosphate, and the RNA product has the attacking phosphate linked to the 5[prime] [Alpha]-phosphate of the catalytic RNA. Thus, reaction with free GDP yields pyrophosphate plus a congener of the eukaryotic message cap (1):

ppG + ppp(5[prime])GRNA [approaches] [pp.sub.i] + G(5[prime])ppp(5[prime])GRNA

The cap structure has been proven by release of the cap from the catalyst, and by comparison with the cap released from an in vitro RNA transcript after initiation with bona fide Gppp[G.sub.OH]. Accordingly, the eukaryotic message cap may have been devised in an RNA world, by RNA catalysts.

The reaction with attacking phosphate nucleophiles can be almost indefinitely generalized (2). The self-capping RNA will react with any nucleoside 3[prime] phosphate, any NMP, any NDP, NTP, or NtetraP to yield caps of varied structure. Even other 5[prime] pRNAs react, though with low yields, to yield unique double molecules with diverging polarity: RNA(5[prime])pp(5[prime])RNA. The attacking nucleophile need not be a nucleotide, because phosphoarginine and phosphogluconate can also cap an RNA catalyst.

Most interestingly, phosphorylated enzyme cofactors react well, yielding RNAs to which the chemical capabilities of (for example) thiamine pyrophosphate or Cofactor A have been precisely linked. These cofactor reactions may be useful in recreating a generation of cofactor-containing RNA catalysts that might have been the progenitors of the modern cofactors themselves.

The self-capping RNA is a metalloenzyme, optimally active in a very simple buffered calcium solution over a broad range of pH. There is an exceptionally strong calcium-binding site, at which barium or strontium (or even magnesium) competes (the ribozyme is inactivated). The evolved capping activity exists side-by-side with the initially selected pyrophosphatase activity (attack of water at the [Alpha]-phosphate, accelerated 1.7 X [10.sup.5]-fold or more (3) over the initial randomized pool), with a cap hydrolysis activity (attack of water, though more slowly, at the [Alpha]-phosphate of the capped RNA), and with a cap exchange activity (attack of a phosphate nucleophile at the [Alpha]-phosphate of an initially capped RNA).

The kinetics of the RNA reactions show some unusual qualities: notably, the sum of the rates of hydrolysis and capping (reaction with phosphates in trans) is a constant whose value is determined by the temperature and phosphate concentration. This points to the formation of an intermediate that can be rapidly captured by either water or an attacking cap. The sum of these two reactions always adds up to the rate at which the 5[prime]-reactive form of the RNA is generated. The nature of this reactive intermediate is unknown at present.

As consequences of the properties of this RNA, both the message cap and the phosphorylated nucleoside cofactors can be plausibly incorporated into accounts of RNA metabolism in an RNA world. Furthermore, the broad substrate specificity of the RNA suggests that it might be made the catalytic subunit in a series of ribozymes, the specificities of which are determined by a second RNA to constitute, for example, a multimeric RNA replicase.

Literature Cited

1. Huang, F., and M. Yarus. 1997. 5[prime] RNA self-capping from guanosine diphosphate. Biochemistry 36: 6557-6563.

2. Huang, F., and M. Yarus. 1997. Versatile 5[prime] phosphoryl coupling of small and large molecules to an RNA. Proc. Nat. Acad. Sci. USA 94: 8965-8969.

3. Huang, F., and M. Yarus. 1997. A calcium-metalloribozyme with autodecapping and pyrophosphatase activities. Biochemistry 36: 14107-14119.

Discussion

BARTEL: It is fascinating that this enzyme, born to hydrolyze its [Alpha], [Beta]-phosphoranhydride, would prefer to catalyze this attack using a phosphate. Is there a way of making sense out of that?

YARUS: Yes. This has to do with the special affinities of calcium and phosphates. I think the Michaelis complex for the capping reactions are phosphates sandwiched between calciums, derived from the site for the original leaving group, pyrophosphate. All of the capping reactions are present because such a complex is there to capture this transition state, which was selected for hydrolysis. The calcium-terminal phosphate complex is crucial to reactivity.

FERRIS: This capping reaction is a common process even without a ribozyme. In all of our reactions we tend to have a chemical reaction on the end of caps of this type as a by-product. This is a general process.

YARUS: Jim (Ferris), you use artificially activated nucleotides. The reaction that you are talking about, cap formation, is not detectable as a spontaneous reaction in our pools. Thus, using activation with the biological leaving group, pyrophosphate, capping is not spontaneous or general.

FERRIS: I would agree. If you were discussing this in an origin of life context, what's artificial and what isn't artificial is a big question. Whether the pyrophosphate or some other leaving group was there is not known. It is also not known if pyrophosphate was actually a prebiotic activating group.

ELLINGTON: You quoted Harold White as saying that these ribonucleotide cofactors exist (1). You then point out that the pyrophosphate linkages in ribo cofactors may be equally fundamental, because they may be vestiges of linkages to ancient catalysts. A prediction from that argument would be that the covalently linked cofactors are going to be better catalysts than the noncovalently linked ones. Normally this would present a problem. The reason for having NAD as a diffusible cofactor rather than nicotinamide amino acid is that one doesn't want it to diffuse. Under your scenario, you can attach the ribozyme, make it a better catalyst, and then it can be de-attached to go and be recycled elsewhere.

YARUS: That's correct. I predict that, in fact, the covalently linked cofactors will be more effective because they are just more precise and more available. RNA and cofactor functionality need not be wasted by putting the cofactors into an enveloping binding site.

Literature Cited

1. White, H. B., III. 1976. Coenzymes as fossils of an earlier metabolic state. J. Mol. Evol. 1: 101-104.
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Title Annotation:includes discussion; Evolution: A Molecular Point of View
Author:Huang, Faqing; Yang, Zhili; Yarus, Mike
Publication:The Biological Bulletin
Date:Jun 1, 1999
Words:1121
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