Early assembly proteins of the large ribosomal subunit of the thermophilic archaebacterium Sulfolobus. Identification and binding to heterologous rRNA species

Studies of ribosome structure in thermophilic archaebacteria may provide valuable information on (i) the mechanisms involved in the stabilization of nucleic acid-protein complexes at high temperatures and (ii) the degree of evolutionary conservation of the ribosomal components in the primary kingdoms of cell descent. In this work we investigate certain aspects of RNA/protein interaction within the large ribosomal subunits of the extremely thermophilic archaebacterium Sulfolobus solfataricus. The ribosomal proteins involved in the early reactions leading to in vitro particle assembly have been identified; it is shown that they can interact with the RNA in a temperature-independent fashion, forming a thermally stable "core" particle that can subsequently be converted into complete 50 S ribosomes. Among the protein components of the core particle, those capable of independently binding to 23 and 5 S RNA species have also been identified. Finally, we show that the early assembly proteins of Sulfolobus large ribosomal subunits are able to interact cooperatively with 23 S RNAs from other archaebacteria or from eubacteria, thereby suggesting that RNA/protein recognition sites are largely conserved within prokaryotic ribosomes. By contrast, no specific binding of the archaebacterial proteins to eukaryotic RNA could be demonstrated.     

Supernumerary proteins of mitochondrial ribosomes

Background

Messenger RNAs encoded by mitochondrial genomes are translated on mitochondrial ribosomes that have unique structure and protein composition compared to prokaryotic and cytoplasmic ribosomes. Mitochondrial ribosomes are a patchwork of core proteins that share homology with prokaryotic ribosomal proteins and new, supernumerary proteins that can be unique to different organisms. In mammals, there are specific supernumerary ribosomal proteins that are not present in other eukaryotes.

Scope of review

Here we discuss the roles of supernumerary proteins in the regulation of mitochondrial gene expression and compare them among different eukaryotic systems. Furthermore, we consider if differences in the structure and organization of mitochondrial genomes may have contributed to the acquisition of mitochondrial ribosomal proteins with new functions.

Major conclusions

The distinct and diverse compositions of mitochondrial ribosomes illustrate the high evolutionary divergence found between mitochondrial genetic systems.

General significance

Elucidating the role of the organism-specific supernumerary proteins may provide a window into the regulation of mitochondrial gene expression through evolution in response to distinct evolutionary paths taken by mitochondria in different organisms. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Mobile domains in ribosomes revealed by proton nuclear magnetic resonance

Ribosomes and subunits from eukaryotic and prokaryotic sources were studied by high-resolution proton magnetic-resonance spectroscopy. If all ribosomal components are firmly bound within the particle, then only broad spectra would be expected. However, relatively sharp resonances were found both in ribosomal subunits and in 70 or 80 S ribosomes. The regions of these mobile protein domains have been partially assigned in Escherichia coli ribosomes. Large and small ribosomal subunits were treated to remove selectively proteins L7/12 and S1, respectively. Sharp proton magnetic resonance spectra were not observed for the stripped large subunit showing that proteins L7/12 comprise the flexible protein region and that there is little other flexibility in the stripped subunit. Complete removal of S1 from the small subunit greatly reduced but did not abolish the sharp protein resonance peaks, indicating that protein S1 contains a substantial flexible component but that other flexible components remain in the stripped small subunit. Evidence for generality of these features of ribosome organization is provided by similar studies on ribosomes from eukaryotic sources.     

Ribosomes from trichomonad protozoa have prokaryotic characteristics.

1. Ribosomes from cells of the genera Trichomonas and Tritrichomonas have been isolated and characterized. The ribosomes from each organism had a sedimentation coefficient of 70S in calibrated sucrose gradients and the subunits sedimented as 50S and 30S particles under the same conditions. 2. The major ribosomal RNAs from each species were identical in size to prokaryotic ribosomal RNAs when examined by denaturing gel electrophoresis. The ribosomes contained both 5.8S and 5S RNAs. 3. The ribosomal proteins were compared by the methods of two-dimensional gel electrophoresis and reversed phase HPLC. Electrophoresis of the ribosomal proteins in two different gel systems indicated the presence of 56 proteins in T. gallinae, 40 in T. bactrachorum and 45 in the Tritrichomonas sp. The protein molecular mass range was 8.5-40 kDa. 4. The HPLC analysis confirmed the protein number established by the gel methods. 5. Both methods of analysis revealed greater similarities between the ribosomal proteins of the 2 Tritrichomonas sp. than between those of the more distantly related T. gallinae and T. bactrachorum.     

Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments

Ribosomal RNAs (rRNAs), assisted by ribosomal proteins, form the basic structure of the ribosome, and play critical roles in protein synthesis. Compared to prokaryotic ribosomes, eukaryotic ribosomes contain elongated rRNAs with several expansion segments and larger numbers of ribosomal proteins. To investigate architectural evolution and functional capability of rRNAs, we employed a Tn5 transposon system to develop a systematic genetic insertion of an RNA segment 31 nt in length into Escherichia coli rRNAs. From the plasmid library harboring a single rRNA operon containing random insertions, we isolated surviving clones bearing rRNAs with functional insertions that enabled rescue of the E. coli strain (Δ7rrn) in which all chromosomal rRNA operons were depleted. We identified 51 sites with functional insertions, 16 sites in 16S rRNA and 35 sites in 23S rRNA, revealing the architecture of E. coli rRNAs to be substantially flexible. Most of the insertion sites show clear tendency to coincide with the regions of the expansion segments found in eukaryotic rRNAs, implying that eukaryotic rRNAs evolved from prokaryotic rRNAs suffering genetic insertions and selections.     

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.     

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.     

Effects of the ribosomal subunit association on the chemical modification of the 16S and 23S RNAs from Escherichia coli

70S ribosomes and 30S and 50S ribosomal subunits from Escherichia coli were modified under non-denaturing conditions with the chemical reagent dimethylsulfate. The ribosomal 23S and 16S RNAs were isolated after the reaction and the last 200 nucleotides from the 3' ends were analyzed for differences in the chemical modification. A number of accessibility changes could be detected for 23S and 16S RNA when 70S ribosomes as opposed to the isolated subunits were modified. In addition to a number of sites which were protected from modification several guanosines showed enhanced reactivities, indicating conformational changes in the ribosomal RNA structures when 30S and 50S subunits associate to a 70S particle. Most of the accessibility changes can be localized in double-helical regions within the secondary structures of the two RNAs. The results confirm the importance of the ribosomal RNAs for ribosomal functions and help to define the RNA domains which constitute the subunit interface of E. coli ribosomes.     

Secondary structure of the Dictyostelium discoideum small subunit ribosomal RNA.

We have used comparative analyses of prokaryotic and eukaryotic small subunit ribosomal RNAs to deduce a secondary structure for the Dictyostelium discoideum 18S rRNA. Most of the duplex regions are evolutionarily conserved in all organisms. We have taken advantage of the variation to the D. discoideum sequence (relative to the yeast and frog 19S rRNAs) to identify additional helical regions which are common to the eukaryotic 18S rRNAs.     

Interaction of the cytoplasmic membrane and ribosomes in Escherichia coli; altered ribosomal proteins in sucrose-dependent spectinomycin-resistant mutants.

Alterations in the ribosomes of sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were studied. Subunit exchange experiments showed that 30S subunits were responsible for the resistance of ribosomes to spectinomycin in all Sucd-Spcr mutants tested. Proteins of 30S ribosomes were analyzed by carboxymethyl cellulose column chromatography based on their elution positions. Mutants YM22 and YM93 had an altered 30S ribosomal protein component, S5, and mutant YM50 had an altered protein, S4. Although a shift of elution position was not detected for all the 30S ribosomal proteins from mutant YM101, the amount of protein S3 was appreciably lowered in the isolated 30S subunits. A partial reconstitution experiment with protein S3 prepared from both the wild-type strain and YM101 revealed that the mutant had altered protein S3 which is responsible for the spectinomycin resistance. These alterations in 30S subunits are discussed in relation to the interaction between ribosomes and the cytoplasmic membrane.     

Comparison of amino acid compositions of mitochondrial and cytoplasmic ribosomal proteins of Saccharomyces cerevisiae.

Summary The amino-acid compositions of the mitochondrial ribosomal subunits of Saccharomyces cerevisiae have been determined and compared to those of cytoplasmic ribosomal subunits. For the large subunits, the mitochondrial and cytoplasmic ribosomes showed major differences in the proportions of arginine, alanine and methionine. For the small subunits, arginine, aspartic acid, alanine, valine and methionine showed marked differences.We have compared these amino-acid compositions with those already published of bacterial and eukaryotic ribosomes by a statistical method of data analysis. It appeared clearly that the yeast mitoribosomes are more distant from bacterial ribosomes than from eukaryotic cytoribosomes.Abbreviations r-proteins ribosomal proteins     

The ribosomal stalk is required for ribosome binding,depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga‐like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the α‐sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the α‐sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild‐type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the ΔP1 and ΔP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild‐type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.     

Generalized structures of the 5S ribosomal RNAs.

The sequences of 5S ribosomal RNAs from a wide-range of organisms have been compared. All sequences fit a generalized 5S RNA secondary structural model. Twenty-three nucleotide positions are found universally, i.e., in 5S RNAs of eukaryotes, prokaryotes, archaebacteria, chloroplasts and mitochondria. One major distinguishing feature between the prokaryotic and eukaryotic 5S RNAs is the number of nucleotide positions between certain universal positions, e.g., prokaryotic 5S RNAs have three positions between the universal positions PuU40 and G44 (using the E. coli numbering system) and eukaryotic 5S RNAs have two. The archaebacterial 5S RNAs appear to resemble the eukaryotic 5S RNAs to varying degrees depending on the species of archaebacteria although all the RNAs conform with the prokaryotic "rule" of chain length between PuU40 and G44. The green plant chloroplast and wheat mitochondrial 5S RNAs appear prokaryotic-like when comparing the number of positions between universal nucleotides. Nucleotide positions common to eukaryotic 5S RNAs have been mapped; in addition, nucleotide sequences, helix lengths and looped-out residues specific to phyla are proposed. Several of the common nucleotides found in the 5S RNAs of metazoan somatic tissue differ in the 5S RNAs of oocytes. These changes may indicate an important functional role of the 5S RNA during oocyte maturation.     

Investigation of structure of the ribosomal L12/P stalk

This review contains recent data on the structure of the functionally important ribosomal domain, L12/P stalk, of the large ribosomal subunit. It is the most mobile site of the ribosome; it has been found in ribosomes of all living cells, and it is involved in the interaction between ribosomes and translation factors. The difference between the structures of the ribosomal proteins forming this protuberance (despite their general resemblance) determines the specificity of interaction between eukaryotic and prokaryotic ribosomes and the respective protein factors of translation. In this review, works on the structures of ribosomal proteins forming the L12/P-stalk in bacteria, archaea, and eukaryotes and data on structural aspects of interactions between these proteins and rRNA are described in detail.     

Analysis of the in vivo assembly pathway of eukaryotic 40S ribosomal proteins

In eukaryotes, in vivo formation of the two ribosomal subunits from four ribosomal RNAs (rRNAs) and approximately 80 ribosomal proteins (r-proteins) involves more than 150 nonribosomal proteins and around 100 small noncoding RNAs. It is temporally and spatially organized within different cellular compartments: the nucleolus, the nucleoplasm, and the cytoplasm. Here, we present a way to analyze how eukaryotic r-proteins of the small ribosomal subunit (SSU) assemble in vivo with rRNA. Our results show that key aspects of the assembly of eukaryotic r-proteins into distinct structural parts of the SSU are similar to the in vitro assembly pathway of their prokaryotic counterparts. We observe that the establishment of a stable assembly intermediate of the eukaryotic SSU body, but not of the SSU head, is closely linked to early rRNA processing events. The formation of assembly intermediates of the head controls efficient nuclear export of the SSU and cytoplasmic pre-rRNA maturation steps.     

Determination of peptide regions on the surface of the eubacterial and archaebacterial ribosome by limited proteolytic digestion.

Limited proteolysis was used in combination with two-dimensional gel electrophoresis, blotting, and amino acid sequence analysis to investigate the surface of intact ribosomal subunits at the peptide and amino acid level. Surface sites of 14 ribosomal proteins from Escherichia coli 50S subunits were determined using proteases with different specificities. To assess the evolutionary conservation of ribosomal topography among eubacteria, large subunits from Bacillus stearothermophilus were also subjected to limited proteolysis. The results obtained indicate a conservation of the three-dimensional ribosomal structure at the peptide level. The data for the eubacterial ribosomes are in full agreement with the model of the 50S protein topography derived from immunological data. Furthermore, peptide surface regions of archaebacterial ribosomes have been investigated. The results presented in this work prove that limited proteolysis can successfully be applied to halophilic and thermophilic ribosomes from archaebacteria.     

Hybrid 80S monomers formed from subunits of ribosomes from protozoa,fungi, plants,and mammals

Free 80S ribosomes of eukaryotic organisms are dissociated by KCl (0.8–1.0 m) in the presence of 2-mercaptoethanol and magnesium ions (10–15mm); the large and small subunits so formed can be recombined to yield 80S monomers. We have now studied the ability of ribosomal subunits from protozoa (Tetrahymena pyriformis), fungi (Allomyces arbuscula, Saccharomyces cerevisiae), plants (pea, wheat), and mammals (rat, mouse, rabbit) to combine to form hybrid ribosomes. In general, both subunits of the species studied participate in the formation of hybrid particles, with the exception of the 60S subunit of Tetrahymena, which does not combine with the small subunit of fungal, plant, or mammalian ribosomes. The interaction of subunits from rat and Tetrahymena ribosomes has been visualized by an electron microscope study of negatively stained preparations. The base sequences of the ribosomal RNAs of these organisms have been compared to those of Saccharomyces by nucleic acid hybridization-competition.This work was supported by a fellowship #PF-529 from the American Cancer Society and by United States Public Health Service, National Institutes of Health grant GM 12449.