Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits

The 18S rRNA of the small eukaryotic ribosomal subunit contains several expansion segments. Electron microscopy data indicate that two of the largest expansion segments are juxtaposed in intact 40S subunits, and data from phylogenetic sequence comparisons indicate that these two expansion segments contain complementary sequences that could form a direct tertiary interaction on the ribosome. We have investigated the secondary structure of the two expansion segments in the region around the putative tertiary interaction. Ribosomes from yeast, wheat, and mouse-three organisms representing separate eukaryotic kingdoms-were isolated, and the structure of ES3 and part of the ES6 region were analyzed using the single-strand-specific chemical reagents CMCT and DMS and the double-strand-specific ribonuclease V1. The modification patterns were analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The investigated sequences were relatively exposed to chemical and enzymatic modification. This is in line with their indicated location on the surface at the solvent side of the subunit. The complementary ES3 and ES6 sequences were clearly inaccessible to single-strand modification, but available for cleavage by double-strand-specific RNase V1. The results are compatible with a direct helical interaction between bases in ES3 and ES6. Almost identical results were obtained with ribosomes from the three organisms investigated.     

Analysis of the secondary structure of expansion segment 39 in ribosomes from fungi, plants and mammals

The structure of expansion segment 39, ES39, in eukaryotic 23 S-like ribosomal RNA was analysed using a combination of chemical and enzymic reagents. Ribosomes were isolated from yeast, wheat, mouse, rat and rabbit, five organisms representing three different eukaryotic kingdoms. The isolated ribosomes were treated with structure-sensitive chemical and enzymic reagents and the modification patterns analysed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The expansion segment was relatively accessible to modification by both enzymic and chemical probes, suggesting that ES39 was exposed on the surface of the ribosomes. The collected modification data were used in secondary structure modelling of the expansion segment. Despite considerable variation in both sequence and length between organisms from different kingdoms, the structure analysis of the expansion segment gave rise to structural fingerprints that allowed identification of homologous structures in ES39 from fungi, plants and mammals. The homologous structures formed an initial helix and an invariant hairpin connected to the initial helix via a long single-stranded loop. The remaining part of the ES39 sequences accounted for most of the length variation seen between the analysed species. This part could form additional, albeit less similar, hairpins. A comparison of ES39 sequences from other fungi, plants and mammals showed that identical structures could be formed in these organisms.     

Proposed secondary structure of eukaryote specific expansion segment 15 in 28S rRNA from mice, rats, and rabbits

The expansion segments in eukaryotic ribosomal RNAs are additional RNA sequences not found in the RNA core common to both prokaryotes and eukaryotes. These regions show large species-dependent variations in sequence and size. This makes it difficult to create secondary structure models for the expansion segments exclusively based on phylogenetic sequence comparison. Here we have used a combination of experimental data and computational methods to generate secondary structure models for expansion segment 15 in 28S rRNA in mice, rats, and rabbits. The experimental data were collected using the structure sensitive reagents DMS, CMCT, kethoxal, micrococcal nuclease, RNase T(1), RNase CL3, RNase V(1), and lead(II) acetate. ES15 was folded with the computer program RNAStructure 3.5 using modification data and phylogenetic similarities between different ES15 sequences. This program uses energy minimization to find the most stable secondary structure of an RNA sequence. The presented secondary structure models include several common structural motifs, but they also have characteristics unique to each organism. Overall, the secondary structure models showed indications of an energetically stable but dynamic structure, easily accessible from the solution by the modification reagents, suggesting that the expansion segment is located on the ribosomal surface.     

Eukaryotic ribosomal proteins lacking a eubacterial counterpart: important players in ribosomal function

The ribosome is a macromolecular machine responsible for protein synthesis in all organisms. Despite the enormous progress in studies on the structure and function of prokaryotic ribosomes, the respective molecular details of the mechanism by which the eukaryotic ribosome and associated factors construct a polypeptide accurately and rapidly still remain largely unexplored. Eukaryotic ribosomes possess more RNA and a higher number of proteins than eubacterial ribosomes. As the tertiary structure and basic function of the ribosomes are conserved, what is the contribution of these additional elements? Elucidation of the role of these components should provide clues to the mechanisms of translation in eukaryotes and help unravel the molecular mechanisms underlying the differences between eukaryotic and eubacterial ribosomes. This article focuses on a class of eukaryotic ribosomal proteins that do not have a eubacterial homologue. These proteins play substantial roles in ribosomal structure and function, and in mRNA binding and nascent peptide folding. The role of these proteins in human diseases and viral expression, as well as their potential use as targets for antiviral agents is discussed.     

Cell-free translation systems from different eukaryotes differ in their sensitivity to a template sugar-phosphate backbone.

To study the role of a messenger sugar-phosphate backbone in the ribosomal decoding process, poly(U) and poly(dT) template activity in different eukaryotic systems has been compared. 80S ribosomes from Saccharomyces cerevisiae appeared to be able to translate poly(dT) both in the presence and in the absence of elongation factors, contrary to poly(U). However, ribosomes from higher eukaryotes (wheat germ, rabbit liver) are completely inefficient in poly(dT) translation. Moreover, rabbit liver ribosomes fail to bind effectively phenylalanyl-tRNA in the presence of poly(dT) although the polynucleotide seems to interact with the ribosomal decoding center. It is also of particular interest that hybrid ribosomes formed from the yeast and rabbit liver subunits can translate poly(dT) only when the large ribosomal subunit from yeast is used.     

Control of ribosome biogenesis in yeast

Ribosome formation in eukaryotes requires the coordinate expression of the genes for about 80 different RNA and protein components. These genes are widely dispersed over the genome, are present in various multiplicities and are transcribed by three different RNA polymerases. Studies in yeast are now beginning to give clues to the molecular mechanisms responsible for control of the production of ribosomal constituents and for their ordered assembly into active ribosomes.     

Deciphering the rules by which dynamics of mRNA secondary structure affect translation efficiency in Saccharomyces cerevisiae

Messenger RNA (mRNA) secondary structure decreases the elongation rate, as ribosomes must unwind every structure they encounter during translation. Therefore, the strength of mRNA secondary structure is assumed to be reduced in highly translated mRNAs. However, previous studies in vitro reported a positive correlation between mRNA folding strength and protein abundance. The counterintuitive finding suggests that mRNA secondary structure affects translation efficiency in an undetermined manner. Here, we analyzed the folding behavior of mRNA during translation and its effect on translation efficiency. We simulated translation process based on a novel computational model, taking into account the interactions among ribosomes, codon usage and mRNA secondary structures. We showed that mRNA secondary structure shortens ribosomal distance through the dynamics of folding strength. Notably, when adjacent ribosomes are close, mRNA secondary structures between them disappear, and codon usage determines the elongation rate. More importantly, our results showed that the combined effect of mRNA secondary structure and codon usage in highly translated mRNAs causes a short ribosomal distance in structural regions, which in turn eliminates the structures during translation, leading to a high elongation rate. Together, these findings reveal how the dynamics of mRNA secondary structure coupling with codon usage affect translation efficiency.     

Structure and evolution of ribosomes

Ribosomes are the only cell organelles occurring in all organisms. E. coli ribosomes, which are the best characterized particles, consist of three RNAs and 53 proteins. All components have been isolated and characterized by chemical, physical and immunological methods. The primary structures of the RNAs and of all the proteins are known. Information about the secondary structure of the proteins derives from circular dichroism measurements and from secondary structure prediction methods. The tertiary structure is being studied by limited proteolysis, proton magnetic resonance and crystallization followed by X-ray analysis. Various methods are being used to elucidate the architecture of the ribosomal particle: three-dimensional image reconstruction of crystals of bacterial ribosomes and/or their subunits; immune electron microscopy; neutron scattering; protein-protein, protein-RNA and RNA-RNA crosslinking; total reconstitution of ribosomal subunits. The results from these studies yield valuable information on the architecture of the ribosomal particle. Many mutants have been isolated in which one or a few ribosomal proteins are altered or even deleted. The genetic and biochemical characterization of these mutants allows conclusions about the importance of these proteins for the function of the ribosome. Ribosomal proteins from various prokaryotic and eukaryotic species have been compared by two-dimensional gel electrophoresis, immunological methods, reconstitution and amino acid sequence analysis. These studies show a strong homology among prokaryotic ribosomal proteins but only a weak homology between proteins from prokaryotic and eukaryotic ribosomes. Comparison of the primary and secondary structures of the ribosomal RNAs from various organisms shows that the secondary structure of the RNA molecules has been strongly conserved throughout evolution.     

Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding

The ribosome, the protein synthesis machinery of the cell, has also been implicated in protein folding. This activity resides within the domain V of the main RNA component of the large subunit of the ribosome. It has been shown that two antiprion drugs 6-aminophenanthridine (6AP) and Guanabenz (GA) bind to the ribosomal RNA and inhibit specifically the protein folding activity of the ribosome. Here, we have characterized with biochemical experiments, the mode of inhibition of these two drugs using ribosomes or ribosomal components active in protein folding (referred to as ’ribosomal folding modulators’ or RFMs) from both bacteria Escherichia coli and yeast Saccharomyces cerevisiae, and human carbonic anhydrase (HCA) as a sample protein. Our results indicate that 6AP and GA inhibit the protein folding activity of the ribosome by competition with the unfolded protein for binding to the ribosome. As a result, the yield of the refolded protein decreases, but the rate of its refolding remains unaffected. Further, 6AP- and GA mediated inhibition of RFM mediated refolding can be reversed by the addition of RFMs in excess. We also demonstrate with delayed addition of the ribosome and the antiprion drugs that there is a short time-span in the range of seconds within which the ribosome interacts with the unfolded protein. Thus we conclude that the protein folding activity of the ribosome is conserved from bacteria to eukaryotes and most likely the substrate for RFMs is an early refolding state of the target protein.     

Ribosome-associated protein LBP/p40 binds to S21 protein of 40S ribosome: analysis using a yeast two-hybrid system

The ribosome-associated protein LBP/p40, which was originally named after "laminin binding protein precursor p40," is distributed on the cell surface as laminin binding protein p67 (LBP/p67), in the nucleus, and on 40S ribosomes. In a broad range of eukaryotes, the localization of LBP/p40 on the 40S ribosome is well conserved. Two yeast homologs of LBP/p40 are believed to be essential for cell viability and each gene product probably corresponds to the assembly and/or stability of the 40S ribosomal subunit. The precise role of LBP/p40 in translation, however, remains to be elucidated, especially in higher eukaryotes. In this report, we used a yeast two-hybrid screening method to isolate molecules associated with human LBP/p40 protein on ribosomes. We found that the 40S ribosomal protein S21 was tightly bound with LBP/p40 in this yeast two-hybrid system and in in vitro analysis. Further, we discovered that the association required a broad region of the LBP/p40 amino acid sequence, which corresponds to the highly conserved region of LBP/p40 homologs among eukaryotes.     

Sequence and secondary structure of the central domain ofDrosophila 26S rRNA: A universal model for the central domain of the large rRNA containing the region in which the central break may happen

Summary An 890-bp sequence from the central region ofDrosophila melanogaster 26S ribosomal DNA (rDNA) has been determined and used in an extensive comparative analysis of the central domain of the large subunit ribosomal RNA (lrRNA) from prokaryotes, organelles, and eukaryotes. An alignment of these different sequences has allowed us to precisely map the regions of the central domain that have highly diverged during evolution. Using this sequence comparison, we have derived a secondary structure model of the central domain ofDrosophila 26S ribosomal RNA (rRNA). We show that a large part of this model can be applied to the central domain of lrRNA from prokaryotes, eukaryotes, and organelles, therefore defining a universal common structural core. Likewise, a comparative study of the secondary structure of the divergent regions has been performed in several organisms. The results show that, despite a nearly complete divergence in their length and sequence, a common structural core is also present in divergent regions. In some organisms, one or two of the divergent regions of the central domain are removed by processing events. The sequence and structure of these regions (fragmentation spacers) have been compared to those of the corresponding divergent regions that remain part of the mature rRNA in other species.     

Dissecting functional similarities of ribosome-associated chaperones from Saccharomyces cerevisiae and Escherichia coli

Ribosome-tethered chaperones that interact with nascent polypeptide chains have been identified in both prokaryotic and eukaryotic systems. However, these ribosome-associated chaperones share no sequence similarity: bacterial trigger factors (TF) form an independent protein family while the yeast machinery is Hsp70-based. The absence of any component of the yeast machinery results in slow growth at low temperatures and sensitivity to aminoglycoside protein synthesis inhibitors. After establishing that yeast ribosomal protein Rpl25 is able to recruit TF to ribosomes when expressed in place of its Escherichia coli homologue L23, the ribosomal TF tether, we tested whether such divergent ribosome-associated chaperones are functionally interchangeable. E. coli TF was expressed in yeast cells that lacked the endogenous ribosome-bound machinery. TF associated with yeast ribosomes, cross-linked to yeast nascent polypeptides and partially complemented the aminoglycoside sensitivity, demonstrating that ribosome-associated chaperones from divergent organisms share common functions, despite their lack of sequence similarity.     

The ribosomal l1 protuberance in yeast is methylated on a lysine residue catalyzed by a seven-beta-strand methyltransferase

Modification of proteins of the translational apparatus is common in many organisms. In the yeast Saccharomyces cerevisiae, we provide evidence for the methylation of Rpl1ab, a well conserved protein forming the ribosomal L1 protuberance of the large subunit that functions in the release of tRNA from the exit site. We show that the intact mass of Rpl1ab is 14 Da larger than its calculated mass with the previously described loss of the initiator methionine residue and N-terminal acetylation. We determined that the increase in mass of yeast Rpl1ab is consistent with the addition of a methyl group to lysine 46 using top-down mass spectrometry. Lysine modification was confirmed by detecting (3)H-N-ε-monomethyllysine in hydrolysates of Rpl1ab purified from yeast cells radiolabeled in vivo with S-adenosyl-l-[methyl-(3)H]methionine. Mass spectrometric analysis of intact Rpl1ab purified from 37 deletion strains of known and putative yeast methyltransferases revealed that only the deletion of the YLR137W gene, encoding a seven-β-strand methyltransferase, results in the loss of the +14-Da modification. We expressed the YLR137W gene as a His-tagged protein in Escherichia coli and showed that it catalyzes N-ε-monomethyllysine formation within Rpl1ab on ribosomes from the ΔYLR137W mutant strain lacking the methyltransferase activity but not from wild-type ribosomes. We also showed that the His-tagged protein could catalyze monomethyllysine formation on a 16-residue peptide corresponding to residues 38-53 of Rpl1ab. We propose that the YLR137W gene be given the standard name RKM5 (ribosomal lysine (K) methyltransferase 5). Orthologs of RKM5 are found only in fungal species, suggesting a role unique to their survival.     

Chemical accessibility of 18S rRNA in native ribosomal complexes: interaction sites of mRNA, tRNA and translation factors

During protein synthesis the ribosome interacts with ligands such as mRNA, tRNA and translation factors. We have studied the effect of ribosome-ligand interaction on the accessibility of 18S rRNA for single strand-specific modification in ribosomal complexes that have been assembled in vivo, i. e. native polysomes. A comparison of the modification patterns derived from programmed and non-programmed ribosomes showed that bases in the 630- and 1060-loops (530- and 790-loops in E. coli) together with two nucleotides in helices 33 and 34 were protected from chemical modification. The majority of the protected sites were homologous to sites previously suggested to be involved in mRNA and/or tRNA binding in prokaryotes and eukaryotes, implying that the interaction sites for these ligands are similar, if not identical, in naturally occurring programmed ribosomes and in in vitro assembled ribosomal complexes. Additional differences between programmed and non-programmed ribosomes were found in hairpin 8. The bases in helix 8 showed increased exposure to chemical modification in the programmed ribosomes. In addition, structural differences in helices 36 and 37 were observed between native 80S run-off ribosomes and 80S ribosomes assembled from isolated 40S and 60S subunits.     

A possible tertiary rRNA interaction between expansion segments ES3 and ES6 in eukaryotic 40S ribosomal subunits

Eukaryotic 16S-like ribosomal RNAs contain 12 so-called expansion segments, i.e., sequences not included in the RNA secondary structure core common to eubacteria, archaea, and eukarya. Two of these expansion segments, ES3 and ES6, are juxtaposed in the recent three-dimensional model of the eukaryotic 40S ribosomal subunit. We have analyzed ES3 and ES6 sequences from more than 2900 discrete eukaryotic species, for possible sequence complementarity between the two expansion segments. The data show that ES3 and ES6 could interact by forming a helix consisting of seven to nine contiguous base pairs in almost all analyzed species. We, therefore, suggest that ES3 and ES6 form a direct RNA-RNA contact in the ribosome.     

rRNA genes from the lower chordate Herdmania momus: structural similarity with higher eukaryotes.

Ascidians, primitive chordates that have retained features of the likely progenitors to all vertebrates, are a useful model to study the evolutionary relationship of chordates to other animals. We have selected the well characterized ribosomal RNA (rRNA) genes to investigate this relationship, and we describe here the cloning and characterization of an entire ribosomal DNA (rDNA) tandem repeat unit from a lower chordate, the ascidian Herdmania momus. rDNA copy number and considerable sequence differences were observed between two H. momus populations. Comparison of rDNA primary sequence and rRNA secondary structures from H. momus with those from other well characterized organisms, demonstrated that the ascidians are more closely related to other chordates than invertebrates. The rDNA tandem repeat makes up a larger percentage (7%) of the genome of this animal than in other higher eukaryotes. The total length of the spacer and transcribed region in H. momus rDNA is small compared to most higher eukaryotes, being less than 8 kb, and the intergenic spacer region consists of smaller internal repeats. Comparative analysis of rDNA sequences has allowed the construction of secondary structures for the 18S, 5.8S and 26S rRNAs.     

Comparative Analysis of the Ribosomal Componentsof the Hydrogenosome-Containing Protist, Trichomonas vaginalis

The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes. In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized. The sedimentation coefficient of the T. vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli. Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80. This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E. coli (approximately 55). N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features. Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T. vaginalis sequences are positioned within a eukaryotic clade. Comparison of deduced secondary structure models of the small and large subunit rRNAs of T. vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T. vaginalis, while variable regions are shortened or lost. These lines of evidence demonstrate that the T. vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type.     

Cross-linking of tobacco mosaic virus RNA and capped polyribonucleotides to 18S rRNA in wheat germ ribosome-mRNA complexes

Tobacco mosaic virus RNA, forming 40S or 80S initiation complexes with wheat germ ribosomes, was covalently bound to 18S ribosomal RNA by the photoreaction with an RNA cross-linking agent, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). Synthetic polyribonucleotide, poly(A, U), with the cap structure m7GpppGmC at the 5'-terminal was also cross-linked to 18S ribosomal RNA in 40S or 80S complexes with ribosomes by the AMT photoreaction. Polyuridylic acid with the same 5'-cap structure, forming 40S complexes but not 80S complexes with ribosomes, was most efficiently cross-linked to 18S ribosomal RNA by the psoralen photoreaction. These results suggest that the interactions between mRNA and 18S rRNA are not necessarily of strict complementarity but occur during formation of the complexes in eukaryotes. The 40S complexes would be then converted to 80S complexes in the presence of the AUG initiation codon or AUG-like triplets containing A and U on the polyribonucleotide chains which interact with 18S ribosomal RNA.