Interactions of the N-terminal domain of ribosomal protein L11 with thiostrepton and rRNA

Ribosomal protein L11 has two domains: the C-terminal domain (L11-C76) binds rRNA, whereas the N-terminal domain (L11-NTD) may variously interact with elongation factor G, the antibiotic thiostrepton, and rRNA. To begin to quantitate these interactions, L11 from Bacillus stearothermophilus has been overexpressed and its properties compared with those of L11-C76 alone in a fluorescence assay for protein-rRNA binding. The assay relies on 2'-amino-butyryl-pyrene-uridine incorporated in a 58-nucleotide rRNA fragment, which gives approximately 15-fold enhancement when L11 or L11-C76 is bound. Although the pyrene tag weakens protein binding, unbiased protein-RNA association constants were obtained in competition experiments with untagged RNA. It was found that (i) intact B. stearothermophilus L11 binds rRNA with K approximately 1.2 x 10(9) m(-1) in buffers with 0.2 m KCl, about 100-fold tighter than Escherichia coli L11; (ii) the N-terminal domain makes a small, salt-dependent contribution to the overall L11-RNA binding affinity (approximately 8-fold enhancement at 0.2 m KCl), (iii) L11 stimulates thiostrepton binding by 2.3 +/- 0.6 x 10(3)-fold, predicting an overall thiostrepton affinity for the ribosome of approximately 10(9) m(-1), and (iv) the yeast homolog of L11 shows no stimulation of thiostrepton binding. The latter observation resolves the question of why eukaryotes are insensitive to the antibiotic. These measurements also show that it is plausible for thiostrepton to compete directly with EF-G.GDP for binding to the L11-RNA complex, and provide a quantitative basis for further studies of L11 function and thiostrepton mechanism.     

NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9.

In addition to the conserved and well-defined RNase H domain, eukaryotic RNases HI possess either one or two copies of a small N-terminal domain. The solution structure of one of the N-terminal domains from Saccharomyces cerevisiae RNase HI, determined using NMR spectroscopy, is presented. The 46 residue motif comprises a three-stranded antiparallel beta-sheet and two short alpha-helices which pack onto opposite faces of the beta-sheet. Conserved residues involved in packing the alpha-helices onto the beta-sheet form the hydrophobic core of the domain. Three highly conserved and solvent exposed residues are implicated in RNA binding, W22, K38 and K39. The beta-beta-alpha-beta-alpha topology of the domain differs from the structures of known RNA binding domains such as the double-stranded RNA binding domain (dsRBD), the hnRNP K homology (KH) domain and the RNP motif. However, structural similarities exist between this domain and the N-terminal domain of ribosomal protein L9 which binds to 23 S ribosomal RNA.     

The three-dimensional structure of the RNA-binding domain of ribosomal protein L2; a protein at the peptidyl transferase center of the ribosome

Ribosomal protein L2 is the largest protein component in the ribosome. It is located at or near the peptidyl transferase center and has been a prime candidate for the peptidyl transferase activity. It binds directly to 23S rRNA and plays a crucial role in its assembly. The three-dimensional structure of the RNA-binding domain of L2 from Bacillus stearothermophilus has been determined at 2.3 A resolution by X-ray crystallography using the selenomethionyl MAD method. The RNA-binding domain of L2 consists of two recurring motifs of approximately 70 residues each. The N-terminal domain (positions 60-130) is homologous to the OB-fold, and the C-terminal domain (positions 131-201) is homologous to the SH3-like barrel. Residues Arg86 and Arg155, which have been identified by mutation experiments to be involved in the 23S rRNA binding, are located at the gate of the interface region between the two domains. The molecular architecture suggests how this important protein has evolved from the ancient nucleic acid-binding proteins to create a 23S rRNA-binding domain in the very remote past.     

All three functional domains of the large ribosomal subunit protein L25 are required for both early and late pre-rRNA processing steps in Saccharomyces cerevisiae

Mutational analysis has shown that the integrity of the region in domain III of 25S rRNA that is involved in binding of ribosomal protein L25 is essential for the production of mature 25S rRNA in the yeast Saccharomyces cerevisiae. However, even structural alterations that do not noticeably affect recognition by L25, as measured by an in vitro assay, strongly reduced 25S rRNA formation by inhibiting the removal of ITS2 from the 27S_B precursor. In order to analyze the role of L25 in yeast pre-rRNA processing further we studied the effect of genetic depletion of the protein or mutation of each of its three previously identified functional domains, involved in nuclear import (N-terminal), RNA binding (central) and 60S subunit assembly (C-terminal), respectively. Depletion of L25 or mutating its (pre-)rRNA-binding domain blocked conversion of the 27S_B precursor to 5.8S/25S rRNA, confirming that assembly of L25 is essential for ITS2 processing. However, mutations in either the N- or the C-terminal domain of L25, which only marginally affect its ability to bind to (pre-)rRNA, also resulted in defective ITS2 processing. Furthermore, in all cases there was a notable reduction in the efficiency of processing at the early cleavage sites A₀, A₁ and A₂. We conclude that the assembly of L25 is necessary but not sufficient for removal of ITS2, as well as for fully efficient cleavage at the early sites. Additional elements located in the N- as well as C-terminal domains of L25 are required for both aspects of pre-rRNA processing.     

Crystal structure of prokaryotic ribosomal protein L9: a bi-lobed RNA-binding protein.

The crystal structure of protein L9 from the Bacillus stearothermophilus ribosome has been determined at 2.8 A resolution using X-ray diffraction methods. This primary RNA-binding protein has a highly elongated and unusual structure consisting of two separated domains joined by a long exposed alpha-helix. Conserved, positively charged and aromatic amino acids on the surfaces of both domains probably represent the sites of specific interactions with 23S rRNA. Comparisons with other prokaryotic L9 sequences show that while the length of the connecting alpha-helix is invariant, the sequence within the exposed central region is not conserved. This suggests that the alpha-helix has an architectural role and serves to fix the relative separation and orientation of the N- and C-terminal domains within the ribosome. The N-terminal domain has structural homology to the smaller ribosomal proteins L7/L12 and L30, and the eukaryotic RNA recognition motif (RRM).     

The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor

The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.     

The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by stabilizing the catalytically active RNA structure. To accomplish this, CYT-18 recognizes conserved structural features of group I intron RNAs using regions of the N-terminal nucleotide-binding fold, intermediate alpha-helical, and C-terminal RNA-binding domains that also function in binding tRNA(Tyr). Curiously, whereas the splicing of the N. crassa mitochondrial large subunit rRNA intron is completely dependent on CYT-18's C-terminal RNA-binding domain, all other group I introns tested thus far are spliced efficiently by a truncated protein lacking this domain. To investigate the function of the C-terminal domain, we used an Escherichia coli genetic assay to isolate mutants of the Saccharomyces cerevisiae mitochondrial large subunit rRNA and phage T4 td introns that can be spliced in vivo by the wild-type CYT-18 protein, but not by the C-terminally truncated protein. Mutations that result in dependence on CYT-18's C-terminal domain include those disrupting two long-range GNRA tetraloop/receptor interactions: L2-P8, which helps position the P1 helix containing the 5'-splice site, and L9-P5, which helps establish the correct relative orientation of the P4-P6 and P3-P9 domains of the group I intron catalytic core. Our results indicate that different structural mutations in group I intron RNAs can result in dependence on different regions of CYT-18 for RNA splicing.     

The RNA binding domains of the nuclear poly(A)-binding protein

The nuclear poly(A)-binding protein (PABPN1) is involved in the synthesis of the mRNA poly(A) tails in most eukaryotes. We report that the protein contains two RNA binding domains, a ribonucleoprotein-type RNA binding domain (RNP domain) located approximately in the middle of the protein sequence and an arginine-rich C-terminal domain. The C-terminal domain also promotes self-association of PABPN1 and moderately cooperative binding to RNA. Whereas the isolated RNP domain binds specifically to poly(A), the isolated C-terminal domain binds non-specifically to RNA and other polyanions. Despite this nonspecific RNA binding by the C-terminal domain, selection experiments show that adenosine residues throughout the entire minimal binding site of approximately 11 nucleotides are recognized specifically. UV-induced cross-links with oligo(A) carrying photoactivatable nucleotides at different positions all map to the RNP domain, suggesting that most or all of the base-specific contacts are made by the RNP domain, whereas the C-terminal domain may contribute nonspecific contacts, conceivably to the same nucleotides. Asymmetric dimethylation of 13 arginine residues in the C-terminal domain has no detectable influence on the interaction of the protein with RNA. The N-terminal domain of PABPN1 is not required for RNA binding but is essential for the stimulation of poly(A) polymerase.     

L11 domain rearrangement upon binding to RNA and thiostrepton studied by NMR spectroscopy

Ribosomal proteins are assumed to stabilize specific RNA structures and promote compact folding of the large rRNA. The conformational dynamics of the protein between the bound and unbound state play an important role in the binding process. We have studied those dynamical changes in detail for the highly conserved complex between the ribosomal protein L11 and the GTPase region of 23S rRNA. The RNA domain is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of the L11 protein (L11_ctd). In addition, the N-terminal domain of L11 (L11_ntd) is implicated in the binding of the natural thiazole antibiotic thiostrepton, which disrupts the elongation factor function. We have studied the conformation of the ribosomal protein and its dynamics by NMR in the unbound state, the RNA bound state and in the ternary complex with the RNA and thiostrepton. Our data reveal a rearrangement of the L11_ntd, placing it closer to the RNA after binding of thiostrepton, which may prevent binding of elongation factors. We propose a model for the ternary L11–RNA–thiostrepton complex that is additionally based on interaction data and conformational information of the L11 protein. The model is consistent with earlier findings and provides an explanation for the role of L11_ntd in elongation factor binding.     

Functional interaction of yeast elongation factor 3 with yeast ribosomes

Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. EF-3 is a monomeric protein with a molecular mass of 116,000. EF-3 is required by yeast ribosomes for in vitro translation and for in vivo growth. The protein stimulates the binding of EF-1 alpha :GTP:aa-tRNA ternary complex to the ribosomal A-site by facilitating release of deacylated-tRNA from the E-site. The reaction requires ATP hydrolysis. EF-3 contains two ATP-binding sequence motifs (NBS). NBSI is sufficient for the intrinsic ATPase function. NBSII is essential for ribosome-stimulated activity. By limited proteolysis, EF-3 was divided into two distinct functional domains. The N-terminal domain lacking the highly charged lysine blocks failed to bind ribosomes and was inactive in the ribosome-stimulated ATPase activity. The C-terminally derived lysine-rich fragment showed strong binding to yeast ribosomes. The purported S5 homology region of EF-3 at the N-terminal end has been reported to interact with 18S ribosomal RNA. We postulate that EF-3 contacts rRNA and/or protein(s) through the C-terminal end. Removal of these residues severely weakens its interaction mediated possibly through the N-terminal domain of the protein.     

Studies of the strand-annealing activity of mammalian hnRNP complex protein A1.

A1 is a major core protein of the mammalian hnRNP complex, and as a purified protein of approximately 34 kDa, A1 is a strong single-stranded nucleic acid binding protein. Several lines of evidence suggest that the protein is organized in discrete domains consisting of an N-terminal segment of approximately 22 kDa and a C-terminal segment of approximately 12 kDa. Each of these domains as a purified fragment is capable of binding to both ssDNA and RNA. We report here that A1 and its C-terminal domain fragment are capable of potent strand-annealing activity for base-pair complementary single-stranded polynucleotides of both RNA and DNA. This effect is not stimulated by ATP. Compared with A1 and the C-terminal fragment, the N-terminal domain fragment has negligible annealing activity. These results indicate that A1 has biochemical activity consistent with a strand-annealing role in relevant reactions, such as pre-mRNA splicing.     

A divergent Sm fold in EDC3 proteins mediates DCP1 binding and P-body targeting

Members of the (L)Sm (Sm and Sm-like) protein family are found across all kingdoms of life and play crucial roles in RNA metabolism. The P-body component EDC3 (enhancer of decapping 3) is a divergent member of this family that functions in mRNA decapping. EDC3 is composed of a N-terminal LSm domain, a central FDF domain, and a C-terminal YjeF-N domain. We show that this modular architecture enables EDC3 to interact with multiple components of the decapping machinery, including DCP1, DCP2, and Me31B. The LSm domain mediates DCP1 binding and P-body localization. We determined the three-dimensional structures of the LSm domains of Drosophila melanogaster and human EDC3 and show that the domain adopts a divergent Sm fold that lacks the characteristic N-terminal α-helix and has a disrupted β4-strand. This domain remains monomeric in solution and lacks several features that canonical (L)Sm domains require for binding RNA. The structures also revealed a conserved patch of surface residues that are required for the interaction with DCP1 but not for P-body localization. The conservation of surface and of critical structural residues indicates that LSm domains in EDC3 proteins adopt a similar fold that has separable novel functions that are absent in canonical (L)Sm proteins.     

NMR structure of bacterial ribosomal protein l20: implications for ribosome assembly and translational control

L20 is a specific protein of the bacterial ribosome, which is involved in the early assembly steps of the 50S subunit and in the feedback control of the expression of its own gene. This dual function involves specific interactions with either the 23S rRNA or its messenger RNA. The solution structure of the free Aquifex aeolicus L20 has been solved. It is composed of an unstructured N-terminal domain comprising residues 1-58 and a C-terminal alpha-helical domain. This is in contrast with what is observed in the bacterial 50S subunit, where the N-terminal region folds as an elongated alpha-helical region. The solution structure of the C-terminal domain shows that several solvent-accessible, conserved residues are clustered on the surface of the molecule and are probably involved in RNA recognition. In vivo studies show that this domain is sufficient to repress the expression of the cistrons encoding L35 and L20 in the IF3 operon. The ability of L20 C-terminal domain to specifically recognise RNA suggests an assembly mechanism for L20 into the ribosome. The pre-folded C-terminal domain would make a primary interaction with a specific site on the 23S rRNA. The N-terminal domain would then fold within the ribosome, participating in its correct 3D assembly.     

The structure of Prp40 FF1 domain and its interaction with the crn-TPR1 motif of Clf1 gives a new insight into the binding mode of FF domains

The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5' end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs.     

The solution structure of REF2-I reveals interdomain interactions and regions involved in binding mRNA export factors and RNA

The RNA binding and export factor (REF) family of mRNA export adaptors are found in several nuclear protein complexes including the spliceosome, TREX, and exon junction complexes. They bind RNA, interact with the helicase UAP56/DDX39, and are thought to bridge the interaction between the export factor TAP/NXF1 and mRNA. REF2-I consists of three domains, with the RNA recognition motif (RRM) domain positioned in the middle. Here we dissect the interdomain interactions of REF2-I and present the solution structure of a functionally competent double domain (NM; residues 1-155). The N-terminal domain comprises a transient helix (N-helix) linked to the RRM by a flexible arm that includes an Arg-rich region. The N-helix, which is required for REF2-I function in vivo, overlaps the highly conserved REF-N motif and, together with the adjacent Arg-rich region, interacts transiently with the RRM. RNA interacts with REF2-I through arginine-rich regions in its N- and C-terminal domains, but we show that it also interacts weakly with the RRM. The mode of interaction is unusual for an RRM since it involves loops L1 and L5. NMR signal mapping and biochemical analysis with NM indicate that DDX39 and TAP interact with both the N and RRM domains of REF2-I and show that binding of these proteins and RNA will favor an open conformation for the two domains. The proximity of the RNA, TAP, and DDX39 binding sites on REF2-I suggests their binding may be mutually exclusive, which would lead to successive ligand binding events in the course of mRNA export.     

Domain I of ribosomal protein L1 is sufficient for specific RNA binding

Ribosomal protein L1 has a dual function as a ribosomal protein binding 23S rRNA and as a translational repressor binding its mRNA. L1 is a two-domain protein with N- and C-termini located in domain I. Earlier it was shown that L1 interacts with the same targets on both rRNA and mRNA mainly through domain I. We have suggested that domain I is necessary and sufficient for specific RNA-binding by L1. To test this hypothesis, a truncation mutant of L1 from Thermus thermophilus, representing domain I, was constructed by deletion of the central part of the L1 sequence, which corresponds to domain II. It was shown that the isolated domain I forms stable complexes with specific fragments of both rRNA and mRNA. The crystal structure of the isolated domain I was determined and compared with the structure of this domain within the intact protein L1. This comparison revealed a close similarity of both structures. Our results confirm our suggestion that in protein L1 its domain I alone is sufficient for specific RNA binding, whereas domain II stabilizes the L1-rRNA complex.