Solution structure of an informationally complex high-affinity RNA aptamer to GTP

Carothers, James M.; Davis, Jonathan H.; Chou, James J.; Szostak, Jack W.

doi:10.1261/rna.2251306

Cited by 66 publications

(75 citation statements)

References 51 publications

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“…For example, amino acid binding by riboswitches employs larger RNA structures which can and do bind more tightly than our selected sites, presumably because riboswitch binding must generate free energy required to drive accompanying regulatory reactions. This is as anticipated-larger RNA sites bind GTP better (Carothers et al 2004) because larger sites are better prestructured for nucleotide binding (Carothers et al 2006), rather than because larger sites make more interactions. Though amino acids are somewhat different double-ended ligands with loose linkage, unlike nucleotides, a similar progression might be anticipated, and has been partially characterized for arginine (Geiger et al 1996).…”

Section: Conclusion For Selected Amino Acid Sitessupporting

confidence: 53%

RNA–Amino Acid Binding: A Stereochemical Era for the Genetic Code

2009

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By combining crystallographic and NMR structural data for RNA-bound amino acids within riboswitches, aptamers, and RNPs, chemical principles governing specific RNA interaction with amino acids can be deduced. Such principles, which we summarize in a ''polar profile'', are useful in explaining newly selected specific RNA binding sites for free amino acids bearing varied side chains charged, neutral polar, aliphatic, and aromatic. Such amino acid sites can be queried for parallels to the genetic code. Using recent sequences for 337 independent binding sites directed to 8 amino acids and containing 18,551 nucleotides in all, we show a highly robust connection between amino acids and cognate coding triplets within their RNA binding sites. The apparent probability (P) that cognate triplets around these sites are unrelated to binding sites is %5.3 9 10 -45 for codons overall, and P % 2.1 9 10 -46 for cognate anticodons. Therefore, some triplets are unequivocally localized near their present amino acids. Accordingly, there was likely a stereochemical era during evolution of the genetic code, relying on chemical interactions between amino acids and the tertiary structures of RNA binding sites. Use of cognate coding triplets in RNA binding sites is nevertheless sparse, with only 21% of possible triplets appearing. Reasoning from such broad recurrent trends in our results, a majority (approximately 75%) of modern amino acids entered the code in this stereochemical era; nevertheless, a minority (approximately 21%) of modern codons and anticodons were assigned via RNA binding sites.A Direct RNA Template scheme embodying a credible early history for coded peptide synthesis is readily constructed based on these observations.

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Section: Conclusion For Selected Amino Acid Sitessupporting

confidence: 53%

RNA–Amino Acid Binding: A Stereochemical Era for the Genetic Code

2009

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“…A subset of these in vitro evolved RNAs have been structurally characterized using NMR or crystallographic techniques [55][56][57][58][59][60]. Relevant to riboswitches, the structure of these RNA aptamers bound to AMP, GTP, FMN, and vitamin B12 have been elucidated (Figure 4).…”

Section: Riboswitches Are Sophisticated Versions Of In Vitro Selectedmentioning

confidence: 99%

“…A comparison of the structural basis for ligand binding of AMP, GTP, FMN, and vitamin B12 aptamers demonstrates this point (compare Figure 2a to e and Figure 4). The recently characterized GTP aptamer serves as the closest mimic of the binding mode of riboswitches [57]. All portions of GTP in this structure are recognized and nearly buried.…”

Section: Riboswitches Are Sophisticated Versions Of In Vitro Selectedmentioning

confidence: 99%

“…This RNA binds ligands with micromolar dissociation constants and promiscuously binds similar molecules with adenine-like moieties including ATP and NAD due to lack of extensive contacts on the solvent exposed side of the ligand. (b) The recently determined GTP aptamer [57] shows the most hallmarks of ligand binding to natural aptamers. The ligand is largely buried in its binding pocket with a K d ~75 nM.…”

Section: Box 1 Apt Usage Of the Term `Aptamer'mentioning

confidence: 99%

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Structural features of metabolite-sensing riboswitches

Wakeman

Winkler

Dann

2007

Trends in Biochemical Sciences

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Riboswitches, metabolite-sensing RNA elements found in untranslated regions of the transcripts they regulate, possess extensive tertiary structure to couple metabolite binding to genetic control. Herein, we discuss recently published structures from four riboswitch classes and compare these natural RNA structures to those of in vitro selected RNA aptamers, which bind similar ligands to those of the riboswitches. Additionally, we examine the glmS riboswitch, the first example of a ribozyme-based riboswitch. This RNA provides the latest twist in the riboswitch field and portends exciting advances in the coming years. Our knowledge of the mechanisms of genetic regulation by riboswitches has increased mightily in recent years and will continue to grow as new riboswitch classes and ligands are discovered and structurally characterized. KeywordsRNA structure; riboswitch; ribozyme; transcription attenuation; noncoding RNAs; posttranscriptional regulation; crystallography; NMR Riboswitches: Remnants of an ancient mode of genetic regulation?The "central dogma" states that information is stored as DNA, transcribed into RNA, and translated into proteins, which are traditionally thought to satisfy most cellular structural and catalytic requirements. Genetic control of these steps is believed to occur primarily through the action of regulatory proteins; however, the importance of noncoding RNAs in these processes has become increasingly appreciated in recent years [1]. In eubacterial organisms, regulatory RNAs can elicit control of gene expression through regulation of transcription attenuation, translation initiation [reviewed in 2,3], or mRNA stability [4]. These eubacterial regulatory RNAs function via diverse intermolecular (trans) or intramolecular (cis) mechanisms to regulate the target mRNA. Trans-acting regulatory RNAs interact with target mRNAs to control translation, mRNA stability, or transcription termination [reviewed in 5,6, 7], whereas others regulate gene expression through specific sequestration of RNA-binding proteins [8]. Cis-acting RNAs, which are the focus of this review, are located within untranslated regions (UTRs) of the mRNA transcript that they regulate. Genetic control by the latter RNA elements can be accomplished either through the sole action of the RNA molecule or in conjunction with recruited protein factors.A classic example of a cis-acting regulatory RNA is the transcription attenuation control of tryptophan biosynthesis genes in certain Gram-negative bacteria [9]. For this mechanism, the kinetics of translation of a short leader peptide dictates the conformational state of the overall 5′-UTR. Under tryptophan-depleted conditions the translating ribosome stalls at tryptophan codons within the leader peptide thereby allowing for formation of an antiterminator helix and [24-26; reviewed in 27,12]. Given their ability to function as sensitive sentinels for intracellular metabolites in a protein-independent manner, these RNAs are likely to require exquisite structural sophistication....

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“…Indeed, our graph-based analysis of random pools (25-100 nucleotides [nt]) showed that different RNA secondary topologies are far from uniformly distributed, with low yields for multiply branched structures, although complex structures gradually become more frequent as RNA length increases (Gevertz et al 2005). Interestingly, recent experimental findings suggest that enhancing the structural diversity of RNA pools increases the possibility of obtaining novel RNAs with high activity (Carothers et al 2004(Carothers et al , 2006. Specifically, GTP aptamers with high-binding affinities are found to be more complex structurally than low-binding-affinity aptamers.…”

Section: Introductionmentioning

confidence: 99%

A computational proposal for designing structured RNA pools for in vitro selection of RNAs

Kim

Gan

Schlick³

2007

RNA

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Although in vitro selection technology is a versatile experimental tool for discovering novel synthetic RNA molecules, finding complex RNA molecules is difficult because most RNAs identified from random sequence pools are simple motifs, consistent with recent computational analysis of such sequence pools. Thus, enriching in vitro selection pools with complex structures could increase the probability of discovering novel RNAs. Here we develop an approach for engineering sequence pools that links RNA sequence space regions with corresponding structural distributions via a ''mixing matrix'' approach combined with a graph theory analysis. We define five classes of mixing matrices motivated by covariance mutations in RNA; these constructs define nucleotide transition rates and are applied to chosen starting sequences to yield specific nonrandom pools. We examine the coverage of sequence space as a function of the mixing matrix and starting sequence via clustering analysis. We show that, in contrast to random sequences, which are associated only with a local region of sequence space, our designed pools, including a structured pool for GTP aptamers, can target specific motifs. It follows that experimental synthesis of designed pools can benefit from using optimized starting sequences, mixing matrices, and pool fractions associated with each of our constructed pools as a guide. Automation of our approach could provide practical tools for pool design applications for in vitro selection of RNAs and related problems.

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Solution structure of an informationally complex high-affinity RNA aptamer to GTP

Cited by 66 publications

References 51 publications

RNA–Amino Acid Binding: A Stereochemical Era for the Genetic Code

RNA–Amino Acid Binding: A Stereochemical Era for the Genetic Code

Structural features of metabolite-sensing riboswitches

A computational proposal for designing structured RNA pools for in vitro selection of RNAs

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