Interactions of ribosomal protein L1 with ribosomal and messenger RNAs

Crystal structures of unbound protein L1 and of its complexes with ribosomal an messenger RNAs are analyzed. It is shown that the values of the apparent association rate constant for L1-RNA depend on conformation of unbound protein L1. It is suggested that L1 binds to rRNA with higher affinity than to mRNA because of additional interactions between domain II of L1 and the loop rRNA region, which is absent in mRNA.     

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.     

New insights into the interaction of ribosomal protein L1 with RNA

The RNA-binding ability of ribosomal protein L1 is of profound interest, since L1 has a dual function as a ribosomal structural protein that binds rRNA and as a translational repressor that binds its own mRNA. Here, we report the crystal structure at 2.6 A resolution of ribosomal protein L1 from the bacterium Thermus thermophilus in complex with a 38 nt fragment of L1 mRNA from Methanoccocus vannielii. The conformation of RNA-bound T.thermophilus L1 differs dramatically from that of the isolated protein. Analysis of four copies of the L1-mRNA complex in the crystal has shown that domain II of the protein does not contribute to mRNA-specific binding. A detailed comparison of the protein-RNA interactions in the L1-mRNA and L1-rRNA complexes identified amino acid residues of L1 crucial for recognition of its specific targets on the both RNAs. Incorporation of the structure of bacterial L1 into a model of the Escherichia coli ribosome revealed two additional contact regions for L1 on the 23S rRNA that were not identified in previous ribosome models.     

The ribosomal binding site for eukaryotic elongation factor EF-2 contains 5 S ribosomal RNA

The possible location of RNA in the ribosomal attachment site for the eukaryotic elongation factor EF-2 was analysed. Stable EF-2 · ribosome complexes formed in the presence of the non-hydrolysable GTP analogue GuoPP[CH₂]P were cross-linked with the short (4 Å between the reactive groups) bifunctional reagent, diepoxybutane. Non-cross-linked EF-2 was removed and the covalent factor-ribosome complex isolated. No interaction between EF-2 and 18 S or 28 S rRNA could be demonstrated. However, density gradient centrifugation of the cross-linked ribosomal complexes showed an increased density (1.25 g/cm³) of the factor, as expected from a covalent complex between EF-2 and a low-molecular-weight RNA species. Treatment of the covalent ribosome-factor complexes with EDTA released approx 50% of the cross-linked EF-2 from the ribosome together with the 5 S rRNA · protein L5 complex. Furthermore, the complex co-migrated with the 5S rRNA · L5 particle in sucrose gradients. Polyacrylamide gel electrophoresis showed that EF-2 was directly linked to 5 S rRNA in the 5 S rRNA · L5 complex, as well as in the complexes isolated by density gradient centrifugation. No traces of 5.8 S rRNA or tRNA could be demonstrated. The data indicate that the ribosomal binding domain for EF-2 contains the 5 S rRNA · protein L5 particle and that EF-2 is located in close proximity to 5 S rRNA within the EF-2 · GuoPP[CH₂]P · ribosome complex.     

A chemical interference study on the interaction of ribosomal protein L11 from Escherichia coli with RNA molecules containing its binding site from 23S rRNA.

The interaction between ribosomal protein L11 from Escherichia coli and in vitro synthesized RNA containing its binding site from 23S rRNA was characterized by identifying nucleotides that interfered with complex formation when chemically modified by diethylpyrocarbonate or hydrazine. Chemically modified RNA was incubated with L11 under conditions appropriate for specific binding of L11 and the resulting protein-RNA complex was separated from unbound RNA on Mg(2+)-containing polyacrylamide gels. The ability to isolate L11 complexes on such gels was affected by the extent of modification by either reagent. Protein-bound and free RNAs were recovered and treated with aniline to identify their content of modified bases. Exclusion of RNA containing chemically altered bases from L11-associated material occurred for 29 modified nucleotides, located throughout the region corresponding to residues 1055-1105 in 23S rRNA. Ten bases within this region did not reproducibly inhibit binding when modified. Multiple bands of RNA were consistently observed on the nondenaturing gels, suggesting that significant intermolecular RNA-RNA interactions had occurred.     

Feedback regulation of ribosomal protein synthesis in E. coli: Localization of the mRNA target sites for repressor action of ribosomal protein L1

E. coli ribosomal protein L1 is a translational repressor of the synthesis in vitro of both proteins encoded in the L11 operon (L11 and L1). L1 is shown to act at a single target site within the first 160 bases of the bicistronic mRNA, near (or at) the translation initiation site of the L11 cistron. Synthesis of L1 apparently requires translation of the preceding L11 cistron, allowing regulation of the synthesis of both proteins from a single mRNA target site. This observation suggests a sequential translation mechanism that results in the equimolar synthesis rates of the two proteins observed in vivo. It was found that the presence of 23S rRNA, but not 16S rRNA, relieves translational inhibition by L1. L1 presumably recognizes structural features of the mRNA target site that are homologous to the L1-binding site of 23S rRNA. Although previous work indicated that translationally inhibited ribosomal protein mRNA is degraded in vivo, L1 repressor action in the present in vitro system was found not to involve mRNA degradation.     

How bacterial ribosomal protein L20 assembles with 23 S ribosomal RNA and its own messenger RNA

In bacteria, the expression of ribosomal proteins is often feedback-regulated at the translational level by the binding of the protein to its own mRNA. This is the case for L20, which binds to two distinct sites of its mRNA that both resemble its binding site on 23 S rRNA. In the present work, we report an NMR analysis of the interaction between the C-terminal domain of L20 (L20C) and both its rRNA- and mRNA-binding sites. Changes in the NMR chemical shifts of the L20C backbone nuclei were used to show that the same set of residues are modified upon addition of either the rRNA or the mRNA fragments, suggesting a mimicry at the atomic level. In addition, small angle x-ray scattering experiments, performed with the rRNA fragment, demonstrated the formation of a complex made of two RNAs and two L20C molecules. A low resolution model of this complex was then calculated using (i) the rRNA/L20C structure in the 50 S context and (ii) NMR and small angle x-ray scattering results. The formation of this complex is interesting in the context of gene regulation because it suggests that translational repression could be performed by a complex of two proteins, each interacting with the two distinct L20-binding sites within the operator.     

Ribosomal protein L1 recognizes the same specific structural motif in its target sites on the autoregulatory mRNA and 23S rRNA

The RNA-binding ability of ribosomal protein L1 is of profound interest since the protein has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding its mRNA. Here, we report the crystal structure of ribosomal protein L1 in complex with a specific fragment of its mRNA and compare it with the structure of L1 in complex with a specific fragment of 23S rRNA determined earlier. In both complexes, a strongly conserved RNA structural motif is involved in L1 binding through a conserved network of RNA–protein H-bonds inaccessible to the solvent. These interactions should be responsible for specific recognition between the protein and RNA. A large number of additional non-conserved RNA–protein H-bonds stabilizes both complexes. The added contribution of these non-conserved H-bonds makes the ribosomal complex much more stable than the regulatory one.     

The ribosomal A site-bound sense and stop codons are similarly positioned towards the A1823-A1824 dinucleotide of the 18S ribosomal RNA

Positioning of the mRNA codon towards the 18S ribosomal RNA in the A site of human 80S ribosomes has been studied applying short mRNA analogs containing either the stop codon UAA or the sense codon UCA with a perfluoroaryl azide group at the uridine residue. Bound to the ribosomal A site, a modified codon crosslinks exclusively to the 40S subunits under mild UV irradiation. This result is inconsistent with the hypothesis [Ivanov et al. (2001) RNA 7, 1683-1692] which requires direct contact between the large rRNA and the stop codon of the mRNA as recognition step at translation termination. Both sense and stop codons crosslink to the same A1823/A1824 invariant dinucleotide in helix 44 of 18S rRNA. The data point to the resemblance between the ternary complexes formed at elongation (sense codon.aminoacyl-tRNA.AA dinucleotide of 18S rRNA) and termination (stop codon.eRF1.AA dinucleotide of 18S rRNA) steps of protein synthesis and support the view that eRF1 may be considered as a functional mimic of aminoacyl-tRNA.     

Cracking pre‐40S ribosomal subunit structure by systematic analyses of RNA–protein cross‐linking

Understanding of eukaryotic ribosome synthesis has been slowed by a lack of structural data for the pre‐ribosomal particles. We report rRNA‐binding sites for six late‐acting 40S ribosome synthesis factors, three of which cluster around the 3′ end of the 18S rRNA in model 3D structures. Enp1 and Ltv1 were previously implicated in ‘beak’ structure formation during 40S maturation—and their binding sites indicate direct functions. The kinase Rio2, putative GTPase Tsr1 and dimethylase Dim1 bind sequences involved in tRNA interactions and mRNA decoding, indicating that their presence is incompatible with translation. The Dim1‐ and Tsr1‐binding sites overlap with those of homologous Escherichia coli proteins, revealing conservation in assembly pathways. The primary binding sites for the 18S 3′‐endonuclease Nob1 are distinct from its cleavage site and were unaltered by mutation of the catalytic PIN domain. Structure probing indicated that at steady state the cleavage site is likely unbound by Nob1 and flexible in the pre‐rRNA. Nob1 binds before pre‐rRNA cleavage, and we conclude that structural reorganization is needed to bring together the catalytic PIN domain and its target.     

Disruption of shape complementarity in the ribosomal protein L1–RNA contact region does not hinder specific recognition of the RNA target site

The formation of a specific and stable complex between two (macro)molecules implies complementary contact surface regions. We used ribosomal protein L1, which specifically binds a target site on 23S rRNA, to study the influence of surface modifications on the protein?RNA affinity. The threonine residue in the universally conserved triad Thr?Met?Gly significant for RNA recognition and binding was substituted by phenylalanine, valine and alanine, respectively. The crystal structure of the mutant Thr217Val of the isolated domain I of L1 from Thermus thermophilus (TthL1) was determined. This structure and that of two other mutants, which had been determined earlier, were analysed and compared with the structure of the wild type L1 proteins. The influence of structural changes in the mutant L1 proteins on their affinity for the specific 23S rRNA fragment was tested by kinetic experiments using surface plasmon resonance (SPR) biosensor analysis. Association rate constants undergo minor changes, whereas dissociation rate constants displayed significantly higher values in comparison with that for the wild type protein. The analysed L1 mutants recognize the specific RNA target site, but the mutant L1?23S rRNA complexes are less stable compared to the wild type complexes. Copyright © 2010 John Wiley & Sons, Ltd.     

Specific interactions of the L10(L12)4 ribosomal protein complex with mRNA, rRNA, and L11

Large ribosomal subunit proteins L10 and L12 form a pentameric protein complex, L10(L12) 4, that is intimately involved in the ribosome elongation cycle. Its contacts with rRNA or other ribosomal proteins have been only partially resolved by crystallography. In Escherichia coli, L10 and L12 are encoded from a single operon for which L10(L12) 4 is a translational repressor that recognizes a secondary structure in the mRNA leader. In this study, L10(L12) 4 was expressed from the moderate thermophile Bacillus stearothermophilus to quantitatively compare strategies for binding of the complex to mRNA and ribosome targets. The minimal mRNA recognition structure is widely distributed among bacteria and has the potential to form a kink-turn structure similar to one identified in the rRNA as part of the L10(L12) 4 binding site. Mutations in equivalent positions between the two sequences have similar effects on L10(L12) 4-RNA binding affinity and identify the kink-turn motif and a loop AA sequence as important recognition elements. In contrast to the larger rRNA structure, the mRNA apparently positions the kink-turn motif and loop for protein recognition without the benefit of Mg (2+)-dependent tertiary structure. The mRNA and rRNA fragments bind L10(L12) 4 with similar affinity ( approximately 10 (8) M (-1)), but fluorescence binding studies show that a nearby protein in the ribosome, L11, enhances L10(L12) 4 binding approximately 100-fold. Thus, mRNA and ribosome targets use similar RNA features, held in different structural contexts, to recognize L10(L12) 4, and the ribosome ensures the saturation of its L10(L12) 4 binding site by means of an additional protein-protein interaction.     

Interferon-mediated effect on ribosomal RNA metabolism

Ribosomal RNA (rRNA) has been shown to be involved in the binding of bacterial messenger RNA (mRNA) and an analogous 18 S rRNA · mRNA complex has been reported in eukaryotic systems. Thus, qualitative changes in host rRNA may be involved in the development of the interferon mediated antiviral state, a process thought to involve the inability of host ribosomes to bind and recognize viral mRNA. Data are reported which suggest that trisomy 21 human fibroblasts respond to human interferon with a marked reduction in cytoplasmic rRNA. [³H]Uridine was used to radioactively label the polysomal RNAs for 24 h beginning 12 h after interferon addition. Subsequent sucrose gradient analysis of the phenol or SDS-extracted RNA revealed that the reduction in radioactive rRNA was nearly complete for the 28 S rRNA. In contrast, considerable residual uridine incorporation was found in the 18 S rRNA species. Corollary data suggesting a net increase in mRNA synthesis and a net decrease in protein synthesis are reported.     

Binding of proteins from the large ribosomal subunits to 5.8 S rRNA of Saccharomyces cerevisiae

Specific binding of purified proteins from the large ribosomal subunits of Saccharomyces cerevisiae to 5.8 S rRNA was examined by three different methods: nitrocellulose membrane filtration, sucrose density gradient centrifugation, and RNA-Sepharose column chromatography. RNA-protein complex formation was proportional to the amount of proteins added to the reaction mixture. The binding of proteins to the RNA could be saturated. Such RNA-protein complexes were isolated on sucrose density gradients. Protein species present in these complexes were isolated, iodinated, and analyzed by two-dimensional polyacrylamide gel electrophoresis. Eleven proteins, L13, L14, L17, L19, L21, L24, L25, L29, L30, L33, and L39, were identified. By comparison, only six proteins interacted with the 5.8 S rRNA-Sepharose under similar ionic conditions. They were proteins L14, L21, L24, L27, L29, and L30. To better characterize these binding proteins, the interaction of individual proteins with 5.8 S rRNA was studied by nitrocellulose membrane filtration. Proteins L14, L19, L21, L29, L33, and L39 were observed to bind individually with 5.8 S rRNA. Binding of each protein to the RNA could be saturated. The apparent association constants (K'a), measured at 4 degrees C and in 30 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 330 mM KCl, and 6 mM beta-mercaptoethanol, ranged from 1.05 to 3.70 X 10(6) M-1.     

Base-paired interaction, in vitro, between hen globin 9S mRNA and eukaryotic ribosomal RNAs.

Hen globin 9S mRNA complexes efficiently with mouse sarcoma 18S rRNA, and to a lesser extent with 28S rRNA, but not with tRNA. The mRNA-18S rRNA complex is dissociated under conditions that lead to disruption of hydrogen bonds, and exhibits a biphasic thermal denaturation curve with Tms at $\text{ca.}$ 39° and 58° in 0.15 $\text{M}$ NaCl-0.03 $\text{M}$ Tris-HCl, pH 7.5. Hen globin mRNA also interacts with 18S rRNAs from various other eukaryotes, and the melting profile and Tm of the complex formed with hen 18S rRNA is very similar to that of the complex formed with mouse sarcoma 18S rRNA.     

RNA chaperone activity of L1 ribosomal proteins: phylogenetic conservation and splicing inhibition

RNA chaperone activity is defined as the ability of proteins to either prevent RNA from misfolding or to open up misfolded RNA conformations. One-third of all large ribosomal subunit proteins from E. coli display this activity, with L1 exhibiting one of the highest activities. Here, we demonstrate via the use of in vitro trans- and cis-splicing assays that the RNA chaperone activity of L1 is conserved in all three domains of life. However, thermophilic archaeal L1 proteins do not display RNA chaperone activity under the experimental conditions tested here. Furthermore, L1 does not exhibit RNA chaperone activity when in complexes with its cognate rRNA or mRNA substrates. The evolutionary conservation of the RNA chaperone activity among L1 proteins suggests a functional requirement during ribosome assembly, at least in bacteria, mesophilic archaea and eukarya. Surprisingly, rather than facilitating catalysis, the thermophilic archaeal L1 protein from Methanococcus jannaschii (MjaL1) completely inhibits splicing of the group I thymidylate synthase intron from phage T4. Mutational analysis of MjaL1 excludes the possibility that the inhibitory effect is due to stronger RNA binding. To our knowledge, MjaL1 is the first example of a protein that inhibits group I intron splicing.     

Domain II of Thermus thermophilus ribosomal protein L1 hinders recognition of its mRNA

The two-domain ribosomal protein L1 has a dual function as a primary rRNA-binding ribosomal protein and as a translational repressor that binds its own mRNA. Here, we report the crystal structure of a complex between the isolated domain I of L1 from the bacterium Thermus thermophilus and a specific mRNA fragment from Methanoccocus vannielii. In parallel, we report kinetic characteristics measured for complexes formed by intact TthL1 and its domain I with the specific mRNA fragment. Although, there is a close similarity between the RNA-protein contact regions in both complexes, the association rate constant is higher in the case of the complex formed by the isolated domain I. This finding demonstrates that domain II hinders mRNA recognition by the intact TthL1.     

The secondary structure of the protein L1 binding region of ribosomal 23S RNA. Homologies with putative secondary structures of the L11 mRNA and of a region of mitochondrial 16S rRNA.

An heterologous complex was formed between E. coli protein L1 and P. vulgaris 23S RNA. We determined the primary structure of the RNA region which remained associated with protein L1 after RNase digestion of this complex. We also identified the loci of this RNA region which are highly susceptible to T1, S1 and Naja oxiana nuclease digestions respectively. By comparison of these results with those previously obtained with the homologous regions of E. coli and B. stearothermophilus 23S RNAs, we postulate a general structure for the protein L1 binding region of bacterial 23S RNA. Both mouse and human mit 16S rRNAs and Xenopus laevis and Tetrahymena 28S rRNAs contain a sequence similar to the E. coli 23s RNS region preceding the L1 binding site. The region of mit 16S rRNA which follows this sequence has a potential secondary structure bearing common features with the L1-associated region of bacterial 23S rRNA. The 5'-end region of the L11 mRNA also has several sequence potential secondary structures displaying striking homologies with the protein L1 binding region of 23S rRNA and this probably explains how protein L1 functions as a translational repressor. One of the L11 mRNA putative structures bears the features common to both the L1-associated region of bacterial 23S rRNA and the corresponding region of mit 16S rRNA.