Solution structure of ψ(32)-modified anticodon stem–loop of Escherichia coli tRNA(Phe)

Nucleoside base modifications can alter the structures and dynamics of RNA molecules and are important in tRNAs for maintaining translational fidelity and efficiency. The unmodified anticodon stem–loop from Escherichia coli tRNA(Phe) forms a trinucleotide loop in solution, but Mg(2+) and dimethylallyl modification of A(37) N6 destabilize the loop-proximal base pairs and increase the mobility of the loop nucleotides. The anticodon arm has three additional modifications, ψ(32), ψ(39), and A(37) C2-thiomethyl. We have used NMR spectroscopy to investigate the structural and dynamical effects of ψ(32) on the anticodon stem-loop from E.coli tRNA(Phe). The ψ(32) modification does not significantly alter the structure of the anticodon stem–loop relative to the unmodified parent molecule. The stem of the RNA molecule includes base pairs ψ(32)-A(38) and U(33)–A(37) and the base of ψ(32) stacks between U(33) and A(31). The glycosidic bond of ψ(32) is in the anti configuration and is paired with A(38) in a Watson–Crick geometry, unlike residue 32 in most crystal structures of tRNA. The ψ(32) modification increases the melting temperature of the stem by ∼3.5°C, although the ψ(32) and U(33) imino resonances are exchange broadened. The results suggest that ψ(32) functions to preserve the stem integrity in the presence of additional loop modifications or after reorganization of the loop into a translationally functional conformation

NMR structure and dynamics of the RNA-binding site for the histone mRNA stem-loop binding protein

The 3' end of replication-dependent histone mRNAs terminate in a conserved sequence containing a stem-loop. This 26-nt sequence is the binding site for a protein, stem-loop binding protein (SLBP), that is involved in multiple aspects of histone mRNA metabolism and regulation. We have determined the structure of the 26-nt sequence by multidimensional NMR spectroscopy. There is a 16-nt stem-loop motif, with a conserved 6-bp stem and a 4-nt loop. The loop is closed by a conserved U.A base pair that terminates the canonical A-form stem. The pyrimidine-rich 4-nt loop, UUUC, is well organized with the three uridines stacking on the helix, and the fourth base extending across the major groove into the solvent. The flanking nucleotides at the base of the hairpin stem do not assume a unique conformation, despite the fact that the 5' flanking nucleotides are a critical component of the SLBP binding site

NMR structure and dynamics of the Specifier Loop domain from the Bacillus subtilis tyrS T box leader RNA

Gram-positive bacteria utilize a tRNA-responsive transcription antitermination mechanism, designated the T box system, to regulate expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes. The RNA transcripts of genes controlled by this mechanism contain 5′ untranslated regions, or leader RNAs, that specifically bind cognate tRNA molecules through pairing of nucleotides in the tRNA anticodon loop with nucleotides in the Specifier Loop domain of the leader RNA. We have determined the solution structure of the Specifier Loop domain of the tyrS leader RNA from Bacillus subtilis. Fifty percent of the nucleotides in the Specifier Loop domain adopt a loop E motif. The Specifier Sequence nucleotides, which pair with the tRNA anticodon, stack with their Watson–Crick edges rotated toward the minor groove and exhibit only modest flexibility. We also show that a Specifier Loop domain mutation that impairs the function of the B. subtilis glyQS T box RNA disrupts the tyrS loop E motif. Our results suggest a mechanism for tRNA–Specifier Loop binding in which the phosphate backbone kink created by the loop E motif causes the Specifier Sequence bases to rotate toward the minor groove, which increases accessibility for pairing with bases in the anticodon loop of tRNA

NMR structure and dynamics of an RNA motif common to the spliceosome branch-point helix and the RNA-binding site for phage GA coat protein

ABSTRACT: The RNA molecules that make up the spliceosome branch-point helix and the binding site for phage GA coat protein share a secondary structure motif in which two consecutive adenine residues occupy the strand opposite a single uridine, creating the potential to form one of two different A‚U base pairs while leaving the other adenine unpaired or bulged. During the splicing of introns out of pre-mRNA, the 2′-OH of the bulged adenine participates in the transesterification reaction at the 5′-exon and forms the branch-point residue of the lariat intermediate. Either adenine may act as the branch-point residue in mammals, but the 3′-proximal adenine does so preferentially. When bound to phage GA coat protein, the bulged adenine loops out of the helix and occupies a binding pocket on the surface of the protein, forming a nucleation complex for phage assembly. The coat protein can bind helices with bulged adenines at either position, but the 3′-proximal site binds with greater affinity. We have studied this RNA motif in a 21 nucleotide hairpin containing a GA coat protein-binding site whose four nucleotide loop has been replaced by a more stable loop from the related phage Ms2. Using heteronuclear NMR spectroscopy, we have determined the structure of this hairpin to an overall precision of 2.0 Å. Both adenine bases stack into the helix, and while all available NOE and coupling constant data are consistent with both possible A‚U base pairs, the base pair involving the 5′-proximal adenine appears to be the major conformation. The 3′-proximal bulged adenine protonates at unusually high pH, and to account for this, we propose a model in which the protonated adenine is stabilized by a hydrogen bond to the uridine O2 of the A‚U base pair. The 2′-OH of the bulged adenine adopts a regular A-form helical geometry, suggesting that in order to participate in the splicing reaction, the conformation of the branch-point helix in the active spliceosome may change from the conformation described here. Thus, while the adenine site preferences of the spliceosome and of phage GA may be due to protein factors, the preferred adenine is predisposed in the free RNA to conformational rearrangement involved in formation of the active complexes. Unpaired or bulged nucleotides are a class of RNA secondary structural motifs that serve as sites of protein recognition and binding (reviewed in ref 1), participate in RNA cleavage and ligation (2, 3), and may play a role in the folding of large RNAs (4). The unique three-dimensional structures of RNAs with unpaired bases presumably contribute to their biological functions. The conformations of bulged bases in DNA oligonucleotides have been extensively studied using nuclear magnetic resonance (NMR) 1 spectroscopy. In general, bulged purine bases tend to stack into the helix (5) while bulged pyrimidine bases either stack into or loop out of the helix depending upon temperature and the identity of neighboring nucleotides (6, 7). Fewer bulged bases in RNA oligonucleotides have been studied, but the conformation of a bulged purine base may depend on whether its flanking regions form Watson-Crick base pairs (8-10). Genetic and biochemical investigations have revealed that a bulged base is necessary for the splicing of introns out of pre-mRNA (3, 11). The first cleavage step of nuclear premRNA splicing involves nucleophilic attack of the phosphate at the 5′-splice site by the 2′-OH group of the branch-point adenine nucleotide to produce the 5′-exon and the lariat intermediat

Comparison of residues 31–39 of () the solution structure of tRNA () and the crystal structures of () fully modified yeast tRNA (,) and () fully modified tRNA (,)

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of ψ-modified anticodon stem–loop of tRNA"</p><p>Nucleic Acids Research 2005;33(22):6961-6971.</p><p>Published online 23 Dec 2005</p><p>PMCID:PMC1322268.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> ψ and A in (A) form a Watson–Crick base pair. In (B) and (C), nucleotides C-A and ψ-C, respectively, form the bifurcated hydrogen bond. The anticodon loop in (A) adopts the tri-loop conformation whereas the anticodon loops in (B) and (C) adopt the U-turn motif. In tRNA, ψ is in the configuration about the glycosidic bond and the loop forms a U-turn motif ()

Stereoview of the superposition of () the stems and () the loops of the 10 converged ψ-ACSL structures

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of ψ-modified anticodon stem–loop of tRNA"</p><p>Nucleic Acids Research 2005;33(22):6961-6971.</p><p>Published online 23 Dec 2005</p><p>PMCID:PMC1322268.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Convergence criteria are given in the text. The views are into the major groove. Only sugar and base heavy atoms are shown and the average r.m.s. deviation for the heavy atoms between the ten structures and the average structure is 1.14 Å. The loop and stem regions are locally well defined, but the propeller twist of the A–U base pair is variable among the structures and slightly increases the r.m.s.d. of the full hairpin

() HNN-COSY and () multiple-bond N-H HSQC spectra showing intra-residue U H5 to N3 and ψ H6 to N1 correlations

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of ψ-modified anticodon stem–loop of tRNA"</p><p>Nucleic Acids Research 2005;33(22):6961-6971.</p><p>Published online 23 Dec 2005</p><p>PMCID:PMC1322268.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The HNN-COSY shows cross-strand H2-N3 crosspeaks for residues 31–39, 38–32, and 37–33 produced by the Watson–Crick base pair configurations. The configuration about the ψ glycosidic bond and participation of ψ N1H in the ψ-A base pair would produce a ψ N1-A H2 crosspeak (dashed circle). The U–A crosspeak is weak and is indicative of a weak hydrogen bond

Sequences of () the ψ-modified and () fully modified RNA hairpins corresponding to the anticodon arm of tRNA

<p><b>Copyright information:</b></p><p>Taken from "Solution structure of ψ-modified anticodon stem–loop of tRNA"</p><p>Nucleic Acids Research 2005;33(22):6961-6971.</p><p>Published online 23 Dec 2005</p><p>PMCID:PMC1322268.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Nucleotide numbering corresponds to the full-length tRNA molecule. ψ designates pseudouridine and msiA designates (2-thiomethyl, N6-dimethylallyl)-adenine. ψ-A base arrangements for () the Watson–Crick base pair and () the bifurcated hydrogen bond interaction