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ScienceWeek
MOLECULAR BIOLOGY: ALLOSTERIC REGULATION OF RNA
The following points are made by Michael Famulok (Science 2004 306:233):
1) Proteins often carry out their cellular functions at a defined time and place, requiring that their activity be precisely regulated. An important mechanism for controlling protein activity is allosteric regulation by a small molecule (i.e., binding of a small molecule to the protein at a location remote from the active site). Binding of this regulatory compound to the allosteric site induces structural rearrangements in the protein that are relayed to the active site, which then becomes either stable or unstable. In allosteric proteins, multiple subunits act cooperatively: Once a regulator is bound to one subunit, another subunit responds with dramatically enhanced affinity, allowing the protein's activity to be modulated as a function of slight changes in the regulator's concentration.
2) Mandal et al (1) have reported that activity regulation by cooperative binding is not restricted to proteins but also is a feature of RNA molecules. They described a bacterial messenger RNA (mRNA) element, a so-called riboswitch, in which two allosteric RNA subunits are triggered cooperatively by the amino acid glycine. This cooperative activation regulates the expression of genes involved in glycine metabolism without the need for any additional proteins.
3) The glycine-responsive riboswitch joins a series of recently identified natural RNA motifs that are controlled by other small-molecule regulators (2). In all of them, the highly selective binding of small molecules to the RNA motifs activates or represses expression of nearby genes by inducing conformational changes in their mRNAs that interfere with transcription or translation. The RNA elements of the new glycine-responsive riboswitch are embedded within the untranslated regions of genes encoding a protein complex that enables bacteria to cleave glycine for consumption as an energy source -- but only if the glycine concentration exceeds a certain level. Accordingly, the expression of the components of the glycine cleavage system must remain in an off-state when the amount of glycine is limited. If not, a resource that is indispensable for vital processes, such as maintenance of protein synthesis, would be invested in energy production, which could just as easily be accomplished with other available molecules.
4) Conversely, if regular fuels such as carbohydrates or fats are scarce, an organism would be at a selective advantage if it could derive energy from sources that are not easily accessible to its competitors. Thus, the glycine riboswitch has to fulfill two important criteria: to act as a genetic "on-switch," and to be able to reliably sense glycine with high specificity within a narrow concentration window. Both of these abilities are far from trivial to achieve even for a protein, and thus set the new riboswitch apart from most other riboswitches. The mechanism of action of the new riboswitch is remarkable and provides a further demonstration of the power of RNA as a regulatory element.
5) Riboswitches are actually natural versions of a class of artificial ligand-binding nucleic acids known as "aptamers". Aptamers were first isolated from complex mixtures of trillions of synthetic sequences by in vitro evolution methods (3,4). They form binding pockets that recognize a huge variety of small organic molecules with high affinity and specificity. In various cases, different aptamer sequences have been identified for the same ligand (5). The glycine riboswitch consists of two different aptamer types that individually bind to a single molecule of glycine.
References (abridged):
1. M. Mandal et al., Science 306, 275 (2004)
2. E. Nudler, A. S. Mironov, Trends Biochem. Sci. 29, 11 (2004)
3. C. Tuerk, L. Gold, Science 249, 505 (1990)
4. A. D. Ellington, J. W. Szostak, Nature 346, 818 (1990)
5. M. Famulok, Curr. Opin. Struct. Biol. 9, 324 (1999)
Science http://www.sciencemag.org
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BIOCHEMISTRY: ON GREEN FLUORESCENT RNA
The following points are made by Michael Famulok (Nature 2004 430:976):
1) Green fluorescent protein (GFP) has proven tremendously useful as a method of creating intense visible fluorescence by entirely molecular biological steps(1). Generally, the gene encoding GFP is fused with a gene of interest so that the resultant protein is tagged with the fluorescent module. It can then be followed in real time by optical imaging to decipher spatio-temporal information on gene expression or protein localization within living cells or tissues(2). Such information is crucial for understanding complex cellular processes that depend on when, where, and how much of a protein is present. The challenge now is to devise similar methods to reveal other molecules involved in cellular functions, such as small-molecule messengers, drugs, metabolites and short functional RNAs. Stojanovic and Kolpashchikov(3) have reported an advance in this area with the development of RNA-based probes that can detect small organic molecules by fluorescence.
2) The new concept relies on aptamers -- short RNA sequences that can bind specifically to particular ligands(4). These molecules are fairly recent discoveries, but already they have been adapted to make probes for various molecules(5). However, current aptamer probes are generated and labelled with fluorescence or radioactivity outside the cell, and a delivery mechanism is needed to get them inside -- a procedure that is potentially disruptive to the very processes under observation. Stojanovic and Kolpashchikov(3) have sidestepped this problem by creating an aptamer probe that can be genetically encoded and produced inside the cell, and that is able to generate marked fluorescence by itself.
3) The basis of their probe is an aptamer that can bind to the dye malachite green (MG). The dye itself is not fluorescent, but its structure is such that when it binds to the aptamer it is forced into a rigid conformation that is highly fluorescent. This, however, is only half the story. Stojanovic and Kolpashchikov's probe has a second module -- another aptamer that binds to whatever small molecule is to be detected.
4) To form the final probe, the authors take advantage of an intrinsic property of aptamers: adaptive binding of ligands. Ever since the first nuclear magnetic resonance structures of aptamer-ligand complexes, it has been clear that the binding of a ligand by an aptamer occurs almost exclusively by adaptive recognition(4). For example, aptamers often contain unpaired loop or bulge regions, which are disordered in the free nucleic acid and only acquire a defined conformation by adaptive folding around the ligand. In the new probe, the MG aptamer and the detector aptamer are linked by a communication module. This module substitutes for one of the helices in the MG-aptamer domain, the formation of which is absolutely required for binding to the dye. When a ligand binds to the detector unit, adaptive binding forces the communication module into a helical structure, providing the right conformation for the MG aptamer to bind to the dye.
References (abridged):
1. Tsien, R. Y. Annu. Rev. Biochem. 67, 509-544 (1998)
2. Zhang, J., Campbell, R. E., Ting, A. Y. & Tsien, R. Y. Nature Rev. Mol. Cell Biol. 3, 906-918 (2002)
3. Stojanovic, M. N. & Kolpashchikov, D. M. J. Am. Chem. Soc. 126, 9266-9270 (2004)
4. Hermann, T. & Patel, D. J. Science 287, 820-825 (2000)
5. Silverman, S. K. RNA 9, 377-383 (2003)
Nature http://www.nature.com/nature
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MOLECULAR BIOLOGY: ON RNA GENE REPRESSION
The following points are made by Thomas R. Cech (Nature 2004 428:263):
1) Organisms profit from synthesizing only those enzymes whose products are in demand. After all, if there's plenty of an amino acid around, why should a cell waste energy making the enzymes that are needed to generate it from precursors? A standard textbook mechanism by which this is achieved is repression. Repressor proteins sense when there is a build-up of a certain product, bind to the gene that encodes the enzyme that generates this product, and thereby inhibit the transcription of more messenger RNA (mRNA). Winkler et al(1) have described a bacterial gene-regulation system with a twist: the repressor is not a protein, but instead is a switchable (on-off) self-cleavage element within the mRNA itself.
2) This newly discovered molecular switch involves an RNA molecule with enzymatic activity. Self-cleavage by this ribozyme is accelerated 1000-fold in the presence of glucosamine-6-phosphate (GlcN6P), a small sugar. GlcN6P is generated by the GlmS enzyme, which is encoded by a portion of the glmS mRNA downstream from the ribozyme sequence. So it is easy to envisage a gene-regulatory circuit in which the glmS mRNA is translated into GlmS protein until the GlcN6P product accumulates; at that point, GlcN6P binds to the special catalytic element in the mRNA, causing it to self-destruct. Although the cleavage event itself leaves the coding region of the message intact, the RNA is either destabilized and subject to degradation, or its ability to be translated into functional protein is compromised.
3) The minimal region of the RNA that can confer this regulatory activity is roughly 75 nucleotides long, the size of a transfer RNA. The chemical mechanism by which it is cleaved during repression has previously been seen in other ribozymes, such as the hammerhead, hairpin, and hepatitis delta virus ribozymes(2), but the structure of its catalytic fold appears to be distinct from these. When placed upstream of an unrelated "reporter" gene, the glmS ribozyme element also repressed its expression, and this required the same sequences that are required for glmS self-cleavage in vitro(1). Thus, the active RNA element is modular and transplantable.
4) Although switching gene expression in this manner is new, there are precedents for the individual components of this regulatory circuit. First, consider GlcN6P binding. Even though it once seemed unlikely that RNA, with its limited diversity of chemical groups and its high negative charge, could specifically bind small molecules, we now know that some naturally occurring RNAs do just that. For example, a particular group of ribozymes forms a pocket that binds guanosine monophosphate, one of the four monomer building-blocks of RNA(3). And a specific region of RNA from the human immunodeficiency virus binds a derivative of the amino acid arginine(4). More recently, short (less than 100 nucleotides) RNA "aptamers" have been identified that specifically bind everything from hydrophobic amino acids to small organic molecules to metal ions(5). In terms of specificity, an RNA aptamer can even distinguish the plant alkaloid theophylline from the closely related molecule caffeine.
References (abridged):
1. Winkler, W. C., Nahvi, A., Roth, A., Collins, J. A. & Breaker, R. R. Nature 428, 281-286 (2004)
2. Doudna, J. A. & Cech, T. R. Nature 418, 222-228 (2002)
3. Michel, F., Hanna, M., Green, R., Bartel, D. P. & Szostak, J. W. Nature 347, 578-580 (1989)
4. Puglisi, J. D. et al. Science 257, 76-80 (1992)
5. Ellington, A. D. & Szostak, J. W. Nature 346, 818-822 (1990)
Nature http://www.nature.com/nature
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