Specific arginine mediated RNA recognition

In many biological systems substantial roles are played by interactions between amino acids and RNA. Among amino acids L-arginine seems to be particularly relevant, because the guanidinium group of arginine side chain can potentially form five hydrogen bonds with appropriately positioned acceptor groups of RNA. Extensive studies reveal that specific arginine recognition is achieved by many different RNAs over a broad range of binding affinities. Arginine is frequently found among amino acids in the nucleic acid-binding motifs in various proteins. For example, specific binding of the HIV-1 Tat protein to its RNA site (TAR) is mediated by a single arginine residue. Free arginine can be also bound by the guanosine site in the group I Tetrahymena ribosomal RNA intron catalytic centre, as well as by numerous RNA motifs, called arginine aptamers, which have been selected in vitro.     

Selection of small molecules by the Tetrahymena catalytic center.

The catalytic center in group I RNAs contains a selective binding site that accommodates both guanosine and L-arginine. In order to understand the specificity of the RNA for small molecules, we analyzed 6 RNAs that vary in this region. Specificity for nucleotides resides substantially in G264 rather than its paired nucleotide C311, and is expressed substantially in Km, with comparatively little variation in kcat. kcat is not notably perturbed even for RNAs with mispairs in the active-site helix. For 5 of 6 sequences, effects of RNA substitutions on arginine binding and GTP reactivity are proportional, confirming that arginine contacts a subset of the groups occupied by G. As a result of particular mutations, reaction with GTP is decreased, and reaction with the natural nucleotides UTP and ATP is enhanced. Molecular modeling of these effects suggests that exceptionally flexible placement of reactants may be an essential quality of RNA-catalyzed splicing. The specificity of the intron can be rationalized by a type of binding model not previously considered, in which the G/arginine site includes adjacent nucleotides (an 'axial' site), rather than a single nucleotide, G264.     

An axial binding site in the Tetrahymena precursor RNA.

Previous studies allow the construction of three distinct models of the binding of G and arginine within the active site of the Tetrahymena self-splicing preribosomal precursor RNA. These models (base triple, axial I and axial II) are now distinguished by measurements on the specificity of RNAs with nucleotide substitutions at positions spanning the site. Because the semi-conserved unpaired nucleotide 263 has no effect on substrate or inhibitor selection by the Tetrahymena RNA we conclude that the axial I model is improbable. In contrast, data with substituted RNAs and nucleoside analogs suggest that nucleotide 265 makes a hydrogen bond with the substrate. Accordingly the active site appears axial because substrate contacts exist at more than one nucleotide on the 5' side of the P7 helix. The effects of this hydrogen bond are observable in cases where the donor or acceptor is on the RNA, whether nucleotide 265 is a purine or pyrimidine, or whether nucleotide 265 is mispaired, wobble paired or normally paired. This pattern is consistent with the axial II model. Molecular dynamics and energy minimization calculations lead to the same conclusions as these site-directed substitutions; the base triple and axial I models are unstable dynamically. Under thermal agitation, the third model site (axial II) is transformed to a related, but more stable structure, axial III. The axial III active site is characterized by the extrusion of the conserved bulged base 263 from the P7 helix, a semi-pocket for G base formed by stacking of nucleotide 262, and formation of all bonds to the G base originally proposed for both the base triple and axial II sites. Because of these hydrogen bonds the axial III site is also consistent with data on enzymatic specificity. The axial III model indicates an unforeseen capacity for pocket formation within the groove of an RNA helix, suggests that the site may be unusually flexible, and bears on a hypothesis concerning the origin of the genetic code.     

The guanosine binding mechanism of the Tetrahymena group I intron.

The Tetrahymena group I intron catalyzes self-splicing through two consecutive transesterification reactions, using a single guanosine-binding site (GBS). In this study, we constructed a model RNA that contains the GBS and a conserved guanosine nucleotide at the 3'-terminus of the intron (omegaG). We determined by NMR the solution structure of this model RNA, and revealed the guanosine binding mechanism of the group I intron. The G22 residue, corresponding to omegaG, participates in a base triple, G22 xx G3 x C12, hydrogen-bonding to the major groove edge of the Watson-Crick G3 x C12 pair. The G22 residue also interacts with A2, which is semi-conserved in all sequenced group I introns.     

Base-pairing potential identified by in vitro selection predicts the kinked RNA backbone observed in the crystal structure of the alfalfa mosaic virus RNA-coat protein complex

The three-dimensional structure of the 3' terminus of alfalfa mosaic virus RNA in complex with an amino-terminal coat protein peptide revealed an unusual RNA fold with inter-AUGC basepairing stabilized by key arginine residues (Guogas, et al., 2004). To probe viral RNA interactions with the full-length coat protein, we have used in vitro genetic selection to characterize potential folding patterns among RNAs isolated from a complex randomized pool. Nitrocellulose filter retention, electrophoretic mobility bandshift analysis, and hydroxyl radical footprinting techniques were used to define binding affinities and to localize the potential RNA-protein interaction sites. Minimized binding sites were identified that included both the randomized domain and a portion of the constant regions of the selected RNAs. The selected RNAs, identified by their ability to bind full-length coat protein, have the potential to form the same unusual inter-AUGC Watson-Crick base pairs observed in the crystal structure, although the primary sequences diverge from the wild-type RNA. A constant feature of both the wild-type RNA and the selected RNAs is a G ribonucleotide in the third position of an AUGC-like repeat. Competitive binding assays showed that substituting adenosine for the constant guanosine in either the wild-type or selected RNAs impaired coat protein binding. These data suggest that the interactions observed in the RNA-peptide structure are likely recapitulated when the full-length protein binds. Further, the results underscore the power of in vitro genetic selection for probing RNA-protein structure and function.     

Small RNA helices as substrates for aminoacylation and their relationship to charging of transfer RNAs.

RNA microhelices that reconstruct the acceptor stems of transfer RNAs can be aminoacylated. The anticodon-independent aminoacylation is sequence-specific and suggests a relationship between amino acids and nucleotide sequences which is different from that of the classical genetic code. The specific aminoacylation of RNA microhelices also suggests a highly differentiated adaptation of the structures of aminoacyl-tRNA synthetases to sequences in the acceptor stems of transfer RNAs.     

Crystal structure of a phage Twort group I ribozyme-product complex

Group I introns are catalytic RNAs capable of orchestrating two sequential phosphotransesterification reactions that result in self-splicing. To understand how the group I intron active site facilitates catalysis, we have solved the structure of an active ribozyme derived from the orf142-I2 intron from phage Twort bound to a four-nucleotide product RNA at a resolution of 3.6 A. In addition to the three conserved domains characteristic of all group I introns, the Twort ribozyme has peripheral insertions characteristic of phage introns. These elements form a ring that completely envelops the active site, where a snug pocket for guanosine is formed by a series of stacked base triples. The structure of the active site reveals three potential binding sites for catalytic metals, and invokes a role for the 2' hydroxyl of the guanosine substrate in organization of the active site for catalysis.     

A phylogenetic analysis reveals an unusual sequence conservation within introns involved in RNA editing

Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosines to inosines within cellular and viral RNAs. Certain glutamate receptor (gluR) pre-mRNAs are substrates for the enzymes in vivo. For example, at the R/G editing site of gluR-B, -C, and -D RNAs, ADARs change an arginine codon (AGA) to a glycine codon (IGA) so that two protein isoforms can be synthesized from a single encoded mRNA; the highly related gluR-A sequence is not edited at this site. To gain insight into what features of an RNA substrate are important for accurate and efficient editing by an ADAR, we performed a phylogenetic analysis of sequences required for editing at the R/G site. We observed highly conserved sequences that were shared by gluR-B, -C, and -D, but absent from gluR-A. Surprisingly, in contrast to results obtained in phylogenetic analyses of tRNA and rRNA, it was the bases in paired, helical regions whose identity was conserved, whereas bases in nonhelical regions varied, but maintained their nonhelical state. We speculate this pattern in part reflects constraints imposed by ADAR's unique specificity and gained support for our hypotheses with mutagenesis studies. Unexpectedly, we observed that some of the gluR introns were conserved beyond the sequences required for editing. The approximately 600-nt intron 13 of gluR-C was particularly remarkable, showing >94% nucleotide identity between human and chicken, organisms estimated to have diverged 310 million years ago.     

Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.     

Solution structure of an RNA fragment with the P7/P9.0 region and the 3'-terminal guanosine of the tetrahymena group I intron

In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 3' terminus of the intron (omegaG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/omegaG) including the GBS and omegaG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, omegaG is recognized by the formation of a base triple with the G264 x C311 base pair, and this recognition is stabilized by the stacking interaction between omegaG and C262. The bulged structure at A263 causes a large helical twist angle (40 +/- 80) between the G264 x C311 and C262 x G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a "Bulge-and-Twist" (BT) pocket. With another twist angle between the C262 x G312 and G413 x C313 base pairs (45 +/- 100), the axis of GBS/omegaG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 A resolution electron density map (Golden et al., Science, 1998, 282:345-358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA.     

Probing the mechanisms of DEAD-box proteins as general RNA chaperones: the C-terminal domain of CYT-19 mediates general recognition of RNA

The DEAD-box protein CYT-19 functions in the folding of several group I introns in vivo and a diverse set of group I and group II RNAs in vitro. Recent work using the Tetrahymena group I ribozyme demonstrated that CYT-19 possesses a second RNA-binding site, distinct from the unwinding active site, which enhances unwinding activity by binding nonspecifically to the adjacent RNA structure. Here, we probe the region of CYT-19 responsible for that binding by constructing a C-terminal truncation variant that lacks 49 amino acids and terminates at a domain boundary, as defined by limited proteolysis. This truncated protein unwinds a six-base-pair duplex, formed between the oligonucleotide substrate of the Tetrahymena ribozyme and an oligonucleotide corresponding to the internal guide sequence of the ribozyme, with near-wild-type efficiency. However, the truncated protein is activated much less than the wild-type protein when the duplex is covalently linked to the ribozyme or single-stranded or double-stranded extensions. Thus, the active site for RNA unwinding remains functional in the truncated CYT-19, but the site that binds the adjacent RNA structure has been compromised. Equilibrium binding experiments confirmed that the truncated protein binds RNA less tightly than the wild-type protein. RNA binding by the compromised site is important for chaperone activity, because the truncated protein is less active in facilitating the folding of a group I intron that requires CYT-19 in vivo. The deleted region contains arginine-rich sequences, as found in other RNA-binding proteins, and may function by tethering CYT-19 to structured RNAs, so that it can efficiently disrupt exposed, non-native structural elements, allowing them to refold. Many other DExD/H-box proteins also contain arginine-rich ancillary domains, and some of these domains may function similarly as nonspecific RNA-binding elements that enhance general RNA chaperone activity.     

The origin of the protein synthesis mechanism

The origin and development of the protein synthesis mechanism is considered in four successive steps. The genetic code is supposed to be controlled by the relative amount (availability) of various amino acids and nucleotides on the one hand, and utility on each amino acid in the polypeptide. on the other hand. Thus, more simple (inutile) and abundant amino acids tended to correspond to codons which were rich in the less frequent base species, G and C. Features of primitive tRNA in the discrimination of amino acid are discussed. Primitive tRNA is proposed to have a discriminator site for amino acid and, separated from it, an anticodon site for interaction with nucleotides. A hypothetical course of subdivision of various nucleic acid species is proposed. In the scheme, mRNA and ribosomal RNA (rRNA) were derived from more primitive insoluble RNA. DNA appeared in the late, not first, step of the development. Several other aspects of evolutionary development of the whole protein synthesis mechanism, e.g., role of the discriminator site on primitive tRNA, modification and subdivision of code catalogue into a more precise specification of amino acids, and possible primordial interactions between tRNA and tRNA-binding sites on insoluble rRNA, are discussed.     

RNA-ligand chemistry: a testable source for the genetic code

In the genetic code, triplet codons and amino acids can be shown to be related by chemical principles. Such chemical regularities could be created either during the code's origin or during later evolution. One such chemical principle can now be shown experimentally. Natural or particularly selected RNA binding sites for at least three disparate amino acids (arginine, isoleucine, and tyrosine) are enriched in codons for the cognate amino acid. Currently, in 517 total nucleotides, binding sites contain 2.4-fold more codon sequences than surrounding nucleotides. The aggregate probability of this enrichment is 10(-7) to 10(-8), had codons and binding site sequences been independent. Thus, at least some primordial coding assignments appear to have exploited triplets from amino acid binding sites as codons.     

RNA Affinity for Molecular L-Histidine; Genetic Code Origins

Selection for affinity for free histidine yields a single RNA aptamer, which was isolated 54 times independently. This RNA is highly specific for the side chain and binds protonated L-histidine with 10²−10³-fold stereoselectivity and a dissociation constant (K_D) of 8–54 μM in different isolates. These histidine-binding RNAs have a common internal loop–hairpin loop structure, based on a conserved RAAGUGGGKKN_0–36 AUGUN_0–2AGKAACAG sequence. Notably, the repetitively isolated sequence contains two histidine anticodons, both implicated by conservation and chemical data in amino acid affinity. This site is probably the simplest structure that can meet our histidine affinity selection, which strengthens experimental support for a “stereochemical” origin of the genetic code.[Reviewing Editor: Niles Lehman]     

Hypothesis: Emergence of Translation as a Result of RNA Helicase Evolution

The origin of translation and the genetic code is one of the major mysteries of evolution. The advantage of templated protein synthesis could have been achieved only when the translation apparatus had already become very complex. This means that the translation machinery, as we know it today, must have evolved towards some different essential function that subsequently sub-functionalised into templated protein synthesis. The hypothesis presented here proposes that translation originated as the result of evolution of a primordial RNA helicase, which has been essential for preventing dying out of the RNA organism in sterile double-stranded form. This hypothesis emerges because modern ribosome possesses RNA helicase activity that likely dates back to the RNA world. I hypothesise that codon-anticodon interactions of tRNAs with mRNA evolved as a mechanism used by RNA helicase, the predecessor of ribosomes, to melt RNA duplexes. In this scenario, peptide bond formation emerged to drive unidirectional movement of the helicase via a molecular ratchet mechanism powered by Brownian motion. I propose that protein synthesis appeared as a side product of helicase activity. The first templates for protein synthesis were functional RNAs (ribozymes) that were unwound by the helicase, and the first synthesised proteins were of random or non-sense sequence. I further suggest that genetic code emerged to avoid this randomness. The initial genetic code thus emerged as an assignment of amino acids to codons according to the sequences of the pre-existing RNAs to take advantage of the side products of RNA helicase function.     

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH₂) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed M_A and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed M_C, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA''s difficulty in specifying a unique conformation and highlighting Nature''s potential to use local transitions of RNA in complex function.     

In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs.

The coat proteins of alfalfa mosaic virus (AMV) and the related ilarviruses bind specifically to the 3' untranslated regions of the viral RNAs, which contain conserved repeats of the tetranucleotide sequence AUGC. The purpose of this study was to develop a more detailed understanding of RNA sequence and/or structural determinants required for coat protein binding by characterizing the role of the AUGC repeats. Starting with a complex pool of 39-nucleotide RNA molecules containing random substitutions in the AUGC repeats, in vitro genetic selection was used to identify RNAs that bound coat protein. After six iterative rounds of selection, amplification, and reselection, 25% of the RNAs selected from the randomized pool were wild type; that is, they contained all four AUGC sequences. Among the 31 clones analyzed, AUGC was clearly the preferred selected sequence at the four repeats, but some nucleotide sequence variability was observed at AUGC(865-868) if the other three AUGC repeats were present. Variant RNAs that bound coat protein with affinities equal to or greater than that of the wild-type molecule were not selected. To extend the in vitro selection results, RNAs containing specific nucleotide substitutions were transcribed in vitro and tested in coat protein and peptide binding assays. The data strongly suggest that the AUGC repeats provide sequence-specific determinants and contribute to a structural platform for specific coat protein binding. Coat protein may function in maintaining the 3' ends of the genomic RNAs during replication by stabilizing an RNA structure that defines the 3' terminus as the initiation site for minus-strand synthesis.