Immunological comparison of ribosomal proteins from archaebacteria.

Antisera were raised in rabbits against ribosomal proteins of Methanobacterium bryantii and used to analyze immunological relationships to ribosomes from other archaebacteria, from eubacteria, and from yeasts. Cross-reaction could be detected within the methanogens and with a member of the extreme halophiles; the degree of immunological similarity reflected the relationship delineated by 16S ribosomal ribonucleic acid oligonucleotide analysis (Fox et al., Science 209:457-463, 1980). With the methods and the anti-total-protein sera employed, there was no detectable cross-reaction with ribosomal proteins or ribosomes from Sulfolobus sp., eubacteria, or yeast.     

Structure, function, and evolution of the family of superoxide dismutase proteins from halophilic archaebacteria.

The protein sequences of seven members of the superoxide dismutase (SOD) family from halophilic archaebacteria have been aligned and compared with each other and with the homologous Mn and Fe SOD sequences from eubacteria and the methanogenic archaebacterium Methanobacterium thermoautotrophicum. Of 199 common residues in the SOD proteins from halophilic archaebacteria, 125 are conserved in all seven sequences, and 64 of these are encoded by single unique triplets. The 74 remaining positions exhibit a high degree of variability, and for almost half of these, the encoding triplets are connected by at least two nonsynonymous nucleotide substitutions. The majority of nucleotide substitutions within the seven genes are nonsynonymous and result in amino acid replacement in the respective protein; silent third-codon-position (synonymous) substitutions are unexpectedly rare. Halophilic SODs contain 30 specific residues that are not found at the corresponding positions of the methanogenic or eubacterial SOD proteins. Seven of these are replacements of highly conserved amino acids in eubacterial SODs that are believed to play an important role in the three-dimensional structure of the protein. Residues implicated in formation of the active site, catalysis, and metal ion binding are conserved in all Mn and Fe SODs. Molecular phylogenies based on parsimony and neighbor-joining methods coherently group the halophile sequences but surprisingly fail to distinguish between the Mn SOD of Escherichia coli and the Fe SOD of M. thermoautotrophicum as the outgroup. These comparisons indicate that as a group, the SODs of halophilic archaebacteria have many unique and characteristic features. At the same time, the patterns of nucleotide substitution and amino acid replacement indicate that these genes and the proteins that they encode continue to be subject to strong and changing selection. This selection may be related to the presence of oxygen radicals and the inter- and intracellular composition and concentration of metal cations.     

Molecular dissection of the silkworm ribosomal stalk complex: the role of multiple copies of the stalk proteins

In animal ribosomes, two stalk proteins P1 and P2 form a heterodimer, and the two dimers, with the anchor protein P0, constitute a pentameric complex crucial for recruitment of translational GTPase factors to the ribosome. To investigate the functional contribution of each copy of the stalk proteins, we constructed P0 mutants, in which one of the two C-terminal helices, namely helix I (N-terminal side) or helix II (C-terminal side) were unable to bind the P1–P2 dimer. We also constructed ‘one-C-terminal domain (CTD) stalk dimers’, P1–P2_ΔC and P1_ΔC–P2, composed of intact P1/P2 monomer and a CTD-truncated partner. Through combinations of P0 and P1–P2 variants, various complexes were reconstituted and their function tested in eEF-2-dependent GTPase and eEF-1α/eEF-2-dependent polyphenylalanine synthesis assays in vitro. Double/single-CTD dimers bound to helix I showed higher activity than that bound to helix II. Despite low polypeptide synthetic activity by a single one-CTD dimer, its binding to both helices considerably increased activity, suggesting that two stalk dimers cooperate, particularly in polypeptide synthesis. This promotion of activity by two stalk dimers was lost upon mutation of the conserved YPT sequence connecting the two helices of P0, suggesting a role for this sequence in cooperativity of two stalk dimers.     

Structure and mechanical properties of the ribosomal L1 stalk three-way junction

The L1 stalk is a key mobile element of the large ribosomal subunit which interacts with tRNA during translocation. Here, we investigate the structure and mechanical properties of the rRNA H76/H75/H79 three-way junction at the base of the L1 stalk from four different prokaryotic organisms. We propose a coarse-grained elastic model and parameterize it using large-scale atomistic molecular dynamics simulations. Global properties of the junction are well described by a model in which the H76 helix is represented by a straight, isotropically flexible elastic rod, while the junction core is represented by an isotropically flexible spherical hinge. Both the core and the helix contribute substantially to the overall H76 bending fluctuations. The presence of wobble pairs in H76 does not induce any increased flexibility or anisotropy to the helix. The half-closed conformation of the L1 stalk seems to be accessible by thermal fluctuations of the junction itself, without any long-range allosteric effects. Bending fluctuations of H76 with a bulge introduced in it suggest a rationale for the precise position of the bulge in eukaryotes. Our elastic model can be generalized to other RNA junctions found in biological systems or in nanotechnology.     

Asymmetric interactions between the acidic P1 and P2 proteins in the Saccharomyces cerevisiae ribosomal stalk.

The Saccharomyces cerevisiae ribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1alpha, P1beta, P2alpha, and P2beta. The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure. Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1alpha, P0-1beta, P0-2alpha, and P0-2beta. The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth. In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition. An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein. The following associations were found: P0-1alpha.P2beta, P0-1beta.P2alpha, P0-2alpha.P1beta, and P0-2beta.P1alpha. These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1alpha.P2beta and P1beta.P2alpha, which have different structural and functional roles.     

Structure and sequence relationships in the lipocalins and related proteins.

The lipocalins and fatty acid-binding proteins (FABPs) are two recently identified protein families that both function by binding small hydrophobic molecules. We have sought to clarify relationships within and between these two groups through an analysis of both structure and sequence. Within a similar overall folding pattern, we find large parts of the lipocalin and FABP structures to be quantitatively equivalent. The three largest structurally conserved regions within the lipocalin common core correspond to characteristic sequence motifs that we have used to determine the constitution of this family using an iterative sequence analysis procedure. This afforded a new interpretation of the family, which highlighted the difficulties of determining a comprehensive and coherent classification of the lipocalins. The first of the three conserved sequence motifs is also common to the FABPs and corresponds to a conserved structural element characteristic of both families. Similarities of structure and sequence within the two families suggests that they form part of a larger "structural superfamily"; we have christened this overall group the calycins to reflect the cup-shaped structure of its members.     

Structure and function of the stalk, a putative regulatory element of the yeast ribosome. Role of stalk protein phosphorylation

The ribosomal stalk is involved directly in the interaction of the elongation factors with the ribosome during protein synthesis. The stalk is formed by a complex of five proteins, four small acidic polypepties and a larger protein which directly interacts with the rRNA at the GTPase center. In eukaryotes, the acidic components correspond to the 12 kDa P1 and P2 proteins, and the RNA binding component is protein P0. All these proteins are found to be phosphorylated in eukaryotic organisms. Previousin vitro data suggested this modification was involved in the activity of this structure. To confirm this possibility a mutational study has shown that phosphorylation takes place at a serine residue close to the carboxyl end of proteins P1, P2 and P0. This serine is part of a consensus casein kinase II phosphorylation site. However, by using a yeast strain carrying a temperature sensitive mutant, it has been shown that CKII is probably not the only enzyme responsible for this modification. Three new protein kinases, RAPI, RAPII and RAPIII, have been purified and compared with CKII and PK60, a previously reported enzyme that phosphorylates the stalk proteins. Differences among the five enzymes have been studied. It has also been found that some typical effects of the PKC kinase stimulate thein vitro phosphorylation of the stalk proteins. All the data available suggest that phosphorylation, although it is not involved in the interaction of the acidic proteins with the ribosome, affects ribosome activity and might participate in some ribosome regulatory mechanism. Presented at theSymposium on Regulation of Translation of Genetic Information by Protein Phosphorylation, 21st Congress of the Czechoslovak Society for Microbiology, Hradec Králové (Czech Republic), September 6–10, 1998.     

Phospholipids and UDP-glucuronosyltransferase. Structure/function relationships

The activation of delipidated microsomal UDP-glucuronosyltransferase from pig liver (GT2P type of enzyme) was studied as a function of several structural modifications of 1-palmitoyl-sn-glycero-3-phosphocholine, which is known to be a good activator of the enzyme. The following types of compounds were tested: substitution of H for OH at position 2; substitution of an ether for an acyl link at position 1; variation of the phosphorus-nitrogen or acyl ester-phosphate ester distances; removal of the glycerol backbone; optical isomers; and substitution of phosphoethanolamine for phosphocholine. Although there were variations in the extent to which these compounds activated delipidated enzyme, all the above types of lipids were effective in this regard. By contrast, lipids with a net negative charge did not activate the enzyme. They inhibited it reversibly. Positively charged lipids, even those lacking a phosphate group, were effective activators. These results indicate that GT2P is unlikely to interact with specific chemical groups of its phospholipid milieu. Effective activation appears instead to depend on the physical properties of the lipid environment.     

Structure and evolution of the L11, L1, L10, and L12 equivalent ribosomal proteins in eubacteria, archaebacteria, and eucaryotes

The genes corresponding to the L11, L1, L10, and L12 equivalent ribosomal proteins (L11e, L1e, L10e, and L12e) of Escherichia coli have been cloned and sequenced from two widely divergent species of archaebacteria, Halobacterium cutirubrum and Sulfolobus solfataricus, and the L10 and four different L12 genes have been cloned and sequenced from the eucaryote Saccharomyces cerevisiae. Alignments between the deduced amino acid sequences of these proteins and to other available homologous proteins of eubacteria and eucaryotes have been made. The data suggest that the archaebacteria are a distinct coherent phylogenetic group. Alignment of the proline-rich L11e proteins reveals that the N-terminal region, believed to be responsible for interaction with release factor 1, is the most highly conserved region and that there is specific conservation of most of the proline residues, which may be important in maintaining the highly elongated structure of the molecule. Although L11 is the most highly methylated protein in the E. coli ribosome, the sites of methylation are not conserved in the archaebacterial L11e proteins. The L1e proteins of eubacteria and archaebacteria show two regions of very high similarity near the center and the carboxy termini of the proteins. The L10e proteins of all kingdoms are colinear and contain approximately three fourths of an L12e protein fused to their carboxy terminus, although much of this fusion has been lost in the truncated eubacterial protein. The archaebacterial and eucaryotic L12e proteins are colinear, whereas the eubacterial protein has suffered a rearrangement through what appear to be gene fusion events. Within the L12e derived region of the L10e proteins there exists a repeated module of 26 amino acids, present in two copies in eucaryotes, three in archaebacteria, and one in eubacteria. This modular sequence is apparently also present in the L12e proteins of all kingdoms and may play a role in L12e dimerization, L10e-L12e complex formation, and the function of the L10e-L12e complex in translation.     

Primary structures of five ribosomal proteins from the archaebacterium Halobacterium marismortui and their structural relationships to eubacterial and eukaryotic ribosomal proteins

The complete amino acid sequences of ribosomal proteins L9, L20, L21/22, L24 and L32 from the archaebacterium Halobacterium marismortui were determined. The comparison of the sequences of these proteins with those from other organisms revealed that proteins L21/22 and L24 are homologous to ribosomal protein Yrp29 from yeast and L19 from rat, respectively, and that H. marismortui L20 is homologous to L30 from eubacteria. H. marismortui ribosomal protein L9 showed sequence homology to both L29 from yeast and L15 from eubacteria. No homologous protein was found for H. marismortui L32. These results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes.     

Structure/function relationships in methylotrophic yeasts

Abstract This symposium marks the 15th anniversary of the discovery of microbodies in methylotrophic yeasts. In the intervening years much has been learned about the structure, function and biogenesis of these organelles and these advances are described. As our endeavours continued, unexpected results have confused commonly held views. This was for instance the case when microbody-minus mutants of yeasts became available which showed that some microbody matrix enzymes may be functional when present in the cytosol while others are not. At the molecular level, our understanding of structure/function relationships is also expanding. Examples are structural elements which relate to protein topogenesis and function of enzymes in different cell compartments. Other, perhaps more unusual, adaptations have also been encountered; some involve protein-protein interactions or even modified cofactors which possibly have helped methylotrophic yeasts to establish and/or maintain themselves in natural ecosystems.     

Structure/function relationships in methylotrophic yeasts

This symposium marks the 15th anniversary of the discovery of microbodies in methylotrophic yeasts. In the intervening years much has been learned about the structure, function and biogenesis of these organelles and these advances are described. As our endeavours continued, unexpected results have confused commonly held views. This was for instance the case when microbody-minus mutants of yeasts became available which showed that some microbody matrix enzymes may be functional when present in the cytosol while others are not. At the molecular level, our understanding of structure/function relationships is also expanding. Examples are structural elements which relate to protein topogenesis and function of enzymes in different cell compartments. Other, perhaps more unusual, adaptations have also been encountered; some involve protein-protein interactions or even modified cofactors which possibly have helped methylotrophic yeasts to establish and/or maintain themselves in natural ecosystems.     

Structure/function relationships in nickel metallobiochemistry

Among the many highlights of nickel metallobiochemistry in 1998 were the discoveries that Escherichia coli glyoxalase I is the first example of a nickel isomerase, and that the superoxide dismutase isolated from Streptomyces seoulensis is a new structural class of superoxide dismutase that features thiolate ligation.     

Assembly of Saccharomyces cerevisiae ribosomal stalk: binding of P1 proteins is required for the interaction of P2 proteins

The yeast ribosomal stalk is formed by a protein pentamer made of the 38 kDa P0 and four 12 kDa acidic P1/P2. The interaction of recombinant acidic proteins P1 alpha and P2 beta with ribosomes from Saccharomyces cerevisiae D4567, lacking all the 12 kDa stalk components, has been used to study the in vitro assembly of this important ribosomal structure. Stimulation of the ribosome activity was obtained by incubating simultaneously the particles with both proteins, which were nonphosphorylated initially and remained unmodified afterward. The N-terminus state, free or blocked, did not affect either the binding or reactivating activity of both proteins. Independent incubation with each protein did not affect the activity of the particles, however, protein P2 beta alone was unable to bind the ribosome whereas P1 alpha could. The binding of P1 alpha alone is a saturable process in acidic-protein-deficient ribosomes and does not take place in complete wild-type particles. Binding of P1 proteins in the absence of P2 proteins takes also place in vivo, when protein P1 beta is overexpressed in S. cerevisiae. In contrast, protein P2 beta is not detected in the ribosome in the P1-deficient D67 strain despite being accumulated in the cytoplasm. The results confirm that neither phosphorylation nor N-terminal blocking of the 12 kDa acidic proteins is required for the assembly and function of the yeast stalk. More importantly, and regardless of the involvement of other elements, they indicate that stalk assembling is a coordinated process, in which P1 proteins would provide a ribosomal anchorage to P2 proteins, and P2 components would confer functionality to the complex.     

Studies on the distribution of ribosomal proteins in mammalian ribosomal subunits.

Murine L5178Y cell ribosomes were dissociated into subunits either with potassium chloride in the presence of puromycin or with the chelating agent EDTA. The proteins of ribosomal subunits obtained by these different methods were compared by means of bidimensional polyacrylamide gel electrophoresis. KCl-derived 60S and 40S subunits were shown to contain 38 and 31 proteins respectively, 3 of them having identical electrophoretic mobilities. Preparations of EDTA-dissociated ribosomal subparticles contained different proportions of these proteins, and 11 major spots were shared between the EDTA-derived large and small ribosomal subunits. Furthermore, 10 proteins absent from subunits treated by high concentrations of KCl were reproducibly found in EDTA-treated ribosomal subparticles.