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Many naturally occurring or artificially created RNAs are capable of binding to guanine or guanine derivatives with high affinity and selectivity. They bind their ligands using very different recognition modes involving a diverse set of hydrogen bonding and stacking interactions. Apparently, the potential structural diversity for guanine, guanosine, and guanine nucleotide binding motifs is far from being fully explored. Szostak and coworkers have derived a large set of different GTP-binding aptamer families differing widely in sequence, secondary structure, and ligand specificity. The so-called class V–GTP aptamer from this set binds GTP with very high affinity and has a complex secondary structure. Here we use solution NMR spectroscopy to demonstrate that the class V aptamer binds GTP through the formation of an intermolecular two-layered G-quadruplex structure that directly incorporates the ligand and folds only upon ligand addition. Ligand binding and G-quadruplex formation depend strongly on the identity of monovalent cations present with a clear preference for potassium ions. GTP binding through direct insertion into an intermolecular G-quadruplex is a previously unobserved structural variation for ligand-binding RNA motifs and rationalizes the previously observed specificity pattern of the class V aptamer for GTP analogs.
DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.
Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes Bowen–Conradi syndrome—a severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910–921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Psi) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-Psi in eukaryotic 18S rRNAs.
The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen–Conradi syndrome (BCS) is caused by a specific Nep1D86G mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Psi). Specific 14C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Psi1191 methylation. ESI MS analysis of acp-modified Psi-nucleosides in a DeltaNep1-mutant showed that Nep1 catalyzes the Psi1191 methylation in vivo. Remarkably, the restored growth of a nep1-1ts mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a deltasnr35 mutant. This strongly suggests a dual Nep1 function, as Psi1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.
Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site.
During translation initiation, the heterotrimeric archaeal translation initiation factor 2 (aIF2) recruits the initiator tRNAi to the small ribosomal subunit. In the stationary growth phase and/or during nutrient stress, Sulfolobus solfataricus aIF2 has a second function: It protects leaderless mRNAs against degradation by binding to their 5′‐ends. The S. solfataricus protein Sso2509 is a translation recovery factor (Trf) that interacts with aIF2 and is responsible for the release of aIF2 from bound mRNAs, thereby enabling translation re‐initiation. It is a member of the domain of unknown function 35 (DUF35) protein family and is conserved in Sulfolobales as well as in other archaea. Here, we present the X‐ray structure of S. solfataricus Trf solved to a resolution of 1.65 Å. Trf is composed of an N‐terminal rubredoxin‐like domain containing a bound zinc ion and a C‐terminal oligosaccharide/oligonucleotide binding fold domain. The Trf structure reveals putative mRNA binding sites in both domains. Surprisingly, the Trf protein is structurally but not sequentially very similar to proteins linked to acyl‐CoA utilization—for example, the Sso2064 protein from S. solfataricus—as well as to scaffold proteins found in the acetoacetyl‐CoA thiolase/high‐mobility group‐CoA synthase complex of the archaeon Methanothermococcus thermolithotrophicus and in a steroid side‐chain‐cleaving aldolase complex from the bacterium Thermomonospora curvata. This suggests that members of the DUF35 protein family are able to act as scaffolding and binding proteins in a wide variety of biological processes.
Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by specific docking domains (DDs), whose structures are not known yet, to synthesize well-defined peptide products. In contrast to classical NRPSs, single-module NRPS subunits responsible for the generation of rhabdopeptide/xenortide-like peptides (RXPs) can act in different order depending on subunit stoichiometry thereby producing peptide libraries. To define the basis for their unusual interaction patterns, we determine the structures of all N-terminal DDs (NDDs) as well as of an NDD-CDD complex and characterize all putative DD interactions thermodynamically for such a system. Key amino acid residues for DD interactions are identified that upon their exchange change the DD affinity and result in predictable changes in peptide production. Recognition rules for DD interactions are identified that also operate in other megasynthase complexes.
