Biomedical subjects

Ronald R Breaker

Publications and source records attributed to Ronald R Breaker.

10 recordsLinked to original sources

PubMed2003

An mRNA structure that controls gene expression by binding S-adenosylmethionine.

Riboswitches are metabolite-binding RNA structures that serve as genetic control elements for certain messenger RNAs. These RNA switches have been identified in all three kingdoms of life and are typically responsible for the control of genes whose protein products are involved in the biosynthesis, transport or utilization of the target metabolite. Herein, we report that a highly conserved RNA domain found in bacteria serves as a riboswitch that responds to the coenzyme S-adenosylmethionine (SAM) with remarkably high affinity and specificity. SAM riboswitches undergo structural reorganization upon introduction of SAM, and these allosteric changes regulate the expression of 26 genes in Bacillus subtilis. This and related findings indicate that direct interaction between small metabolites and allosteric mRNAs is an important and widespread form of genetic regulation in bacteria.

Bacillus subtilis↗

PubMed2003

Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria.

Riboswitches are metabolite binding domains within certain messenger RNAs that serve as precision sensors for their corresponding targets. Allosteric rearrangement of mRNA structure is mediated by ligand binding, and this results in modulation of gene expression. We have identified a class of riboswitches that selectively recognizes guanine and becomes saturated at concentrations as low as 5 nM. In Bacillus subtilis, this mRNA motif is located on at least five separate transcriptional units that together encode 17 genes that are mostly involved in purine transport and purine nucleotide biosynthesis. Our findings provide further examples of mRNAs that sense metabolites and that control gene expression without the need for protein factors. Furthermore, it is now apparent that riboswitches contribute to the regulation of numerous fundamental metabolic pathways in certain bacteria.

5' Untranslated Regions↗

PubMed2003

Substrate specificity and reaction kinetics of an X-motif ribozyme.

The X-motif is an in vitro-selected ribozyme that catalyzes RNA cleavage by an internal phosphoester transfer reaction. This ribozyme class is distinguished by the fact that it emerged as the dominant clone among at least 12 different classes of ribozymes when in vitro selection was conducted to favor the isolation of high-speed catalysts. We have examined the structural and kinetic properties of the X-motif in order to provide a framework for its application as an RNA-cleaving agent and to explore how this ribozyme catalyzes phosphoester transfer with a predicted rate constant that is similar to those exhibited by the four natural self-cleaving ribozymes. The secondary structure of the X-motif includes four stem elements that form a central unpaired junction. In a bimolecular format, two of these base-paired arms define the substrate specificity of the ribozyme and can be changed to target different RNAs for cleavage. The requirements for nucleotide identity at the cleavage site are GD, where D = G, A, or U and cleavage occurs between the two nucleotides. The ribozyme has an absolute requirement for a divalent cation cofactor and exhibits kinetic behavior that is consistent with the obligate binding of at least two metal ions.

Base Sequence↗

PubMed2003

Metabolite-binding RNA domains are present in the genes of eukaryotes.

Genetic control by metabolite-binding mRNAs is widespread in prokaryotes. These riboswitches are typically located in noncoding regions of mRNA, where they selectively bind their target compound and subsequently modulate gene expression. We have identified mRNA elements in fungi and in plants that match the consensus sequence and structure of thiamine pyrophosphate-binding domains of prokaryotes. In Arabidopsis, the consensus motif resides in the 3'-UTR of a thiamine biosynthetic gene, and the isolated RNA domain binds the corresponding coenzyme in vitro. These results suggest that metabolite-binding mRNAs are possibly involved in eukaryotic gene regulation and that some riboswitches might be representatives of an ancient form of genetic control.

Animals↗

PubMed2003

A common speed limit for RNA-cleaving ribozymes and deoxyribozymes.

It is widely believed that the reason proteins dominate biological catalysis is because polypeptides have greater chemical complexity compared with nucleic acids, and thus should have greater enzymatic power. Consistent with this hypothesis is the fact that protein enzymes typically exhibit chemical rate enhancements that are far more substantial than those achieved by natural and engineered ribozymes. To investigate the true catalytic power of nucleic acids, we determined the kinetic characteristics of 14 classes of engineered ribozymes and deoxyribozymes that accelerate RNA cleavage by internal phosphoester transfer. Half approach a maximum rate constant of approximately 1 min(-1), whereas ribonuclease A catalyzes the same reaction approximately 80,000-fold faster. Additional biochemical analyses indicate that this commonly encountered ribozyme "speed limit" coincides with the theoretical maximum rate enhancement for an enzyme that uses only two specific catalytic strategies. These results indicate that ribozymes using additional catalytic strategies could be made that promote RNA cleavage with rate enhancements that equal those of proteins.

Catalysis↗

PubMed2003

Ribozyme speed limits.

The speed at which RNA molecules decompose is a critical determinant of many biological processes, including those directly involved in the storage and expression of genetic information. One mechanism for RNA cleavage involves internal phosphoester transfer, wherein the 2'-oxygen atom carries out an SN2-like nucleophilic attack on the adjacent phosphorus center (transesterification). In this article, we discuss fundamental principles of RNA transesterification and define a conceptual framework that can be used to assess the catalytic power of enzymes that cleave RNA. We deduce that certain ribozymes and deoxyribozymes, like their protein enzyme counterparts, can bring about enormous rate enhancements.

Esterification↗

PubMed2002

An mRNA structure that controls gene expression by binding FMN.

The RFN element is a highly conserved domain that is found frequently in the 5'-untranslated regions of prokaryotic mRNAs that encode for flavin mononucleotide (FMN) biosynthesis and transport proteins. We report that this domain serves as the receptor for a metabolite-dependent riboswitch that directly binds FMN in the absence of proteins. Our results also indicate that in Bacillus subtilis, the riboswitch most likely controls gene expression by causing premature transcription termination of the ribDEAHT operon and precluding access to the ribosome-binding site of ypaA mRNA. Sequence and structural analyses indicate that the RFN element is a natural FMN-binding aptamer, the allosteric character of which is harnessed to control gene expression.

5' Untranslated Regions↗

PubMed2002

Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression.

Although proteins fulfil most of the requirements that biology has for structural and functional components such as enzymes and receptors, RNA can also serve in these capacities. For example, RNA has sufficient structural plasticity to form ribozyme and receptor elements that exhibit considerable enzymatic power and binding specificity. Moreover, these activities can be combined to create allosteric ribozymes that are modulated by effector molecules. It has also been proposed that certain messenger RNAs might use allosteric mechanisms to mediate regulatory responses depending on specific metabolites. We report here that mRNAs encoding enzymes involved in thiamine (vitamin B(1)) biosynthesis in Escherichia coli can bind thiamine or its pyrophosphate derivative without the need for protein cofactors. The mRNA-effector complex adopts a distinct structure that sequesters the ribosome-binding site and leads to a reduction in gene expression. This metabolite-sensing regulatory system provides an example of a 'riboswitch' whose evolutionary origin might pre-date the emergence of proteins.

Allosteric Regulation↗

PubMed2002

Engineered allosteric ribozymes as biosensor components.

RNA and DNA molecules can be engineered to function as molecular switches that trigger catalytic events when a specific target molecule becomes bound. Recent studies on the underlying biochemical properties of these constructs indicate that a significant untapped potential exists for the practical application of allosteric nucleic acids. Engineered molecular switches can be used to report the presence of specific analytes in complex mixtures, making possible the creation of new types of biosensor devices and genetic control elements.

Allosteric Regulation↗

PubMed2002

Genetic control by a metabolite binding mRNA.

Messenger RNAs are typically thought of as passive carriers of genetic information that are acted upon by protein- or small RNA-regulatory factors and by ribosomes during the process of translation. We report that the 5'-untranslated sequence of the Escherichia coli btuB mRNA assumes a more proactive role in metabolic monitoring and genetic control. The mRNA serves as a metabolite-sensing genetic switch by selectively binding coenzyme B(12) without the need for proteins. This binding event establishes a distinct RNA structure that is likely to be responsible for inhibition of ribosome binding and consequent reduction in synthesis of the cobalamin transport protein BtuB. This finding, along with related observations, supports the hypothesis that metabolic monitoring through RNA-metabolite interactions is a widespread mechanism of genetic control.

5' Untranslated Regions↗