The social life of ribosomal proteins

Ribosomal proteins hold a unique position in biology because their function is so closely tied to the large rRNAs of the ribosomes in all kingdoms of life. Following the determination of the complete crystal structures of both the large and small ribosomal subunits from bacteria, the functional role of the proteins has often been overlooked when focusing on rRNAs as the catalysts of translation. In this review we highlight some of the many known and important functions of ribosomal proteins, both during translation on the ribosome and in a wider context.     

Analysis, by two-dimensional gel electrophoresis, of ribosome crystal proteins

A ribosome crystal is an aggregate of ribosomes which are packed in a regular array. Preliminary experiments analysing the proteins from ribosome crystals by two-dimensional gel electrophoresis show that, although most proteins appear similar to those from polyribosomes, four extra proteins also seem to be characteristic of ribosome crystals.     

A cradle for new proteins: trigger factor at the ribosome

Newly synthesized proteins leave the ribosome through a narrow tunnel in the large subunit. During ongoing synthesis, nascent protein chains are particularly sensitive to aggregation and degradation because they emerge from the ribosome in an unfolded state. In bacteria, the first protein to interact with nascent chains and facilitate their folding is the ribosome-associated chaperone trigger factor. Recently, crystal structures of trigger factor and of its ribosome-binding domain in complex with the large ribosomal subunit revealed that the chaperone adopts an extended 'dragon-shaped' fold with a large hydrophobic cradle, which arches over the exit of the ribosomal tunnel and shields newly synthesized proteins. These structural results, together with recent biochemical data on trigger factor and its interplay with other chaperones and factors that interact with the nascent chain, provide a comprehensive view of the role of trigger factor during co-translational protein folding.     

Short communications.

A ribosome crystal is an aggregate of ribosomes which are packed in a regular array. Preliminary experiments analysing the proteins from ribosome crystals by two-dimensional gel electrophoresis show that, although most proteins appear similar to those from polyribosomes, four extra proteins also seem to be characteristic of ribosome crystals.     

Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn⁻ phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn⁻ phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn⁻ phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes.     

The crystal structure of saporin SO6 from Saponaria officinalis and its interaction with the ribosome

The 2.0 Å resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented. The fold typical of other plant toxins is conserved, despite some differences in the loop regions. The loop between strands β7 and β8 in the C-terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate. Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications. A contact surface inside the C-terminal region of saporin has been identified. Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition.     

Crystal Structure of the Yeast Ribosomal Protein rpS3 in Complex with Its Chaperone Yar1

Eukaryotic ribosome assembly involves a plethora of factors, which ensure that a correctly folded ribosome contains all ribosomal protein components. Among these assembly factors, Yar1 has recently emerged as a molecular chaperone for ribosomal protein rpS3 of the small ribosomal subunit (40S) in yeast. In complex with its chaperone, rpS3 is imported into the nucleus and protected from aggregation. How rpS3 and other ribosomal proteins are initially sequestered and subsequently integrated into pre-ribosomal particles is currently poorly understood. Here, we present the crystal structure of yeast rpS3 in complex with its chaperone Yar1 at 2.8 Å resolution. The crystal structure rationalizes how Yar1 can protect rpS3 from aggregation while facilitating nuclear import and suggests a mechanism for a stepwise exchange of molecular partners that ribosomal proteins interact with during ribosome assembly.     

Atomic model of the Thermus thermophilus 70S ribosome developed in silico

The ribosome is a large molecular complex that consists of at least three ribonucleic acid molecules and a large number of proteins. It translates genetic information from messenger ribonucleic acid and makes protein accordingly. To better understand ribosomal function and provide information for designing biochemical experiments require knowledge of the complete structure of the ribosome. For expanding the structural information of the ribosome, we took on the challenge of developing a detailed Thermus thermophilus ribosomal structure computationally. By combining information derived from the low-resolution x-ray structure of the 70S ribosome (providing the overall fold), high-resolution structures of the ribosomal subunits (providing the local structure), sequences, and secondary structures, we have developed an atomic model of the T. thermophilus ribosome using a homology modeling approach. Our model is stereochemically sound with a consistent single-species sequence. The overall folds of the three ribosomal ribonucleic acids in our model are consistent with those in the low-resolution crystal structure (root mean-square differences are all <1.9 Å). The large overall interface area (~2500 Ų) of intersubunit bridges B2a, B3, and B5, and the inherent flexibility in regions connecting the contact residues are consistent with these bridges serving as anchoring patches for the ratcheting and rolling motions between the two subunits during translocation.     

Polymorphism in fowl serum albumin. VII. Distribution and activity of free and membrane-bound polysomes in developing fowl liver

The quantity and activities of membrane-bound and free polysomes in livers from chick embryos at successive stages of development were compared in cell-free protein-synthesizing systems. Membrane-bound polysomes increased 2-fold between 8 and 18 days of development, while total ribosome content remained constant. Free polysome activity also remained constant during this period, while that of membrane-bound (total--free) polysomes decreased, possibly because of an increase in ribonuclease activity in this fraction. Serum albumin biosynthesis occurred primarily on membrane-bound polysomes. With liver development, increased secretion of serum proteins may be correlated with synthesis of serum albumin on increasing numbers of membrane bound polyribosomes.     

Ribosomal protein L6: structural evidence of gene duplication from a primitive RNA binding protein.

In all cells, protein synthesis is coordinated by the ribosome, a large ribonucleoprotein particle that is composed of > 50 distinct protein molecules and several large RNA molecules. Here we present the crystal structure of ribosomal protein L6 from the thermophilic bacterium Bacillus stearothermophilus solved at 2.6 A resolution. L6 contains two domains with almost identical folds, implying that it was created by an ancient gene duplication event. The surface of the molecule displays several likely sites of interaction with other components of the ribosome. The RNA binding sites appear to be localized in the C-terminal domain whereas the N-terminal domain contains the potential sites for protein-protein interactions. The domain structure is homologous with several other ribosomal proteins and to a large family of eukaryotic RNA binding proteins.     

Proteomic characterization of the Chlamydomonas reinhardtii chloroplast ribosome. Identification of proteins unique to th e70 S ribosome

We have conducted a proteomic analysis of the 70 S ribosome from the Chlamydomonas reinhardtii chloroplast. Twenty-seven orthologs of Escherichia coli large subunit proteins were identified in the 50 S subunit, as well as an ortholog of the spinach plastid-specific ribosomal protein-6. Several of the large subunit proteins of C. reinhardtii have short extension or insertion sequences, but overall the large subunit proteins are very similar to those of spinach chloroplast and E. coli. Two proteins of 38 and 41 kDa, designated RAP38 and RAP41, were identified from the 70 S ribosome that were not found in either of the ribosomal subunits. Phylogenetic analysis identified RAP38 and RAP41 as paralogs of spinach CSP41, a chloroplast RNA-binding protein with endoribonuclease activity. Overall, the chloroplast ribosome of C. reinhardtii is similar to those of spinach chloroplast and E. coli, but the C. reinhardtii ribosome has proteins associated with the 70 S complex that are related to non-ribosomal proteins in other species. In addition, the 30 S subunit contains unusually large orthologs of E. coli S2, S3, and S5 and a novel S1-type protein (Yamaguchi, K. et al., (2002) Plant Cell 14, 2957-2974). These additional proteins and domains likely confer functions used to regulate chloroplast translation in C. reinhardtii.     

A small protein unique to bacteria organizes rRNA tertiary structure over an extensive region of the 50 S ribosomal subunit

A number of small, basic proteins penetrate into the structure of the large subunit of the ribosome. While these proteins presumably aid in the folding of the rRNA, the extent of their contribution to the stability or function of the ribosome is unknown. One of these small, basic proteins is L36, which is highly conserved in Bacteria, but is not present in Archaea or Eucarya. Comparison of ribosome crystal structures shows that the space occupied by L36 in a bacterial ribosome is empty in an archaeal ribosome. To ask what L36 contributes to ribosome stability and function, we have constructed an Escherichia coli strain lacking ribosomal protein L36; cell growth is slowed by 40-50% between 30 degrees C and 42 degrees C. Ribosomes from this deletion strain sediment normally and have a full complement of proteins, other than L36. Chemical protection experiments comparing rRNA from wild-type and L36-deficient ribosomes show the expected increase in reagent accessibility in the immediate vicinity of the L36 binding site, but suggest that a cooperative network of rRNA tertiary interactions has been disrupted along a path extending 60 A deep into the ribosome. These data argue that L36 plays a significant role in organizing 23 S rRNA structure. Perhaps the Archaea and Eucarya have compensated for their lack of L36 by maintaining more stable rRNA tertiary contacts or by adopting alternative protein-RNA interactions elsewhere in the ribosome.     

Identification of ribosomal protein autoantigens

Approximately 20% of patients with systemic lupus erythematosus and with anti-Sm autoantibodies synthesize autoantibodies, called anti-rRNP, to components of the ribosome. We found that anti-rRNP sera reacted predominantly with three ribosomal phosphoproteins of approximate Mr = 38,000, 16,000 and 15,000, both by immunoprecipitation and by immunoblotting. The human autoantibodies cross-reacted with similar antigens present in rodent, brine shrimp, and yeast cells but reacted weakly if at all with proteins of bacteria. Thus the human autoantibodies recognize epitopes that are widely conserved in evolution. Purified ribosomal proteins together with specific rabbit antisera were used to identify the two smaller rRNP antigens as the acidic phosphoproteins of the large ribosomal subunit, designated P1/P2(L40/L41) (rat), eL7/eL12 (Artemia, brine shrimp), and A1/A2 (yeast). These proteins function in the elongation step of protein synthesis in an analogous fashion to the L7/L12 ribosomal proteins of E. coli. The 38,000-dalton rRNP antigen corresponds to a nonacidic protein also associated with the large ribosomal subunit. The human autoantibodies appear to have a specificity similar to that of a previously described mouse monoclonal antibody obtained from mice injected with heterologous (chick) ribosomes, suggesting that both the human polyclonal autoantibodies and the mouse monoclonal recognize a class of epitope(s) that is common in all three ribosomal proteins. In addition, we found that many of the anti-ribosomal sera contained a further class of autoantibodies reactive with naked RNA. These may be similar to the anti-RNA antibodies previously described in both humans and mice with autoimmune disease.     

栝楼籽核糖体失活蛋白的纯化、性质及晶体生长研究

栝楼（Ｔｒｉｃｈｏｓａｎｔｈｅｓｋｉｒｉｌｏｗｉ）籽经粉碎抽提、硫酸铵沉淀、阳离子交换及凝胶过滤柱层析等步骤,得到一种单链核糖体失活蛋白－Ｔｒｉｃｈｏｋｉｒｉｎ（ＴＣＫ）．ＳＤＳ－ＰＡＧＥ和ＩＥＦ显示为单一条带,其分子量为２９ｋＤ,ｐＩ≥９．３,含糖量约为１．７５％．该蛋白对兔网织红细胞裂解液系统的蛋白质合成具较强的抑制活性,ＩＣ５０为６．７×１０－１０ｍｏｌ／Ｌ．改进了纯化方法,提高了产率,并培养出晶体．     

The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation

Background

Defects in the human Shwachman-Bodian-Diamond syndrome (SBDS) protein-coding gene lead to the autosomal recessive disorder characterised by bone marrow dysfunction, exocrine pancreatic insufficiency and skeletal abnormalities. This protein is highly conserved in eukaryotes and archaea but is not found in bacteria. Although genomic and biophysical studies have suggested involvement of this protein in RNA metabolism and in ribosome biogenesis, its interacting partners remain largely unknown.

Results

We determined the crystal structure of the SBDS orthologue from Methanothermobacter thermautotrophicus (mthSBDS). This structure shows that SBDS proteins are highly flexible, with the N-terminal FYSH domain and the C-terminal ferredoxin-like domain capable of undergoing substantial rotational adjustments with respect to the central domain. Affinity chromatography identified several proteins from the large ribosomal subunit as possible interacting partners of mthSBDS. Moreover, SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments, combined with electrophoretic mobility shift assays (EMSA) suggest that mthSBDS does not interact with RNA molecules in a sequence specific manner.

Conclusion

It is suggested that functional interactions of SBDS proteins with their partners could be facilitated by rotational adjustments of the N-terminal and the C-terminal domains with respect to the central domain. Examination of the SBDS protein structure and domain movements together with its possible interaction with large ribosomal subunit proteins suggest that these proteins could participate in ribosome function.     

Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome

SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the heterotrimeric SecYEG complex, in a so-called posttranslational manner. A recent study further showed binding of a monomeric state of SecA to the ribosome. However, the true oligomeric state of SecA remains controversial because SecA can also form functional dimers, and high-resolution crystal structures exist for both the monomer and the dimer. Here we present the cryo-electron microscopy structures of Escherichia coli SecA bound to the ribosome. We show that not only a monomeric SecA binds to the ribosome but also that two copies of SecA can be observed that form an elongated dimer. Two copies of SecA completely surround the tunnel exit, providing a unique environment to the nascent polypeptides emerging from the ribosome. We identified the N-terminal helix of SecA required for a stable association with the ribosome. The structures indicate a possible function of the dimeric form of SecA at the ribosome.     

Exchange and stability of HeLa ribosomal proteins in vivo.

The relative stabilities of individual HeLa ribosomal proteins and their capacity for exchange between ribosome-bound and -free states in the cytoplasm were examined. Most ribosomal proteins on cytoplasmic ribosomes were found to have uniform, high stability as measured by comparing the short term (12-hour) to steady state (3-day) labeling ratios determined for each ribosomal protein. This would be expected if the proteins in ribosomes either were all stable or were all degraded as a unit. The data do not rule out the possibility that individual proteins have different stabilities prior to their assembly into ribosomes. Four proteins labeled atypically. One large subunit protein (L5) had a lower than average ratio. We interpret this low ratio as being due to a large free pool of this protein. Three proteins (L10, L28, S2) had higher than average ratios, interpreted as being due to reduced protein stability. Two of these proteins (L10, L28) with high ratios were also found to exchange in vivo. The exchangeable proteins may be subject to increased degradation during the time that they spend in the exchangeable free pool. The third protein (S2) with an atypically high ratio is thought to be degraded or altered while on the ribosome, or slowly lost as ribosomes age, because exchange of this protein was not detected. These interpretations and some alternate interpretations are explained. The exchange of three large subunit proteins (L10, L19, L28) was detected by labeling of protein after ribosome synthesis had been inhibited with actinomycin D. Autoradiography of two-dimensional polyacrylamide gels showed labeling of these spots.     

Crystal structure of the ribosomal protein S6 from Thermus thermophilus.

The amino acid sequence and crystal structure of the ribosomal protein S6 from the small ribosomal subunit of Thermus thermophilus have been determined. S6 is a small protein with 101 amino acid residues. The 3D structure, which was determined to 2.0 A resolution, consists of a four-stranded anti-parallel beta-sheet with two alpha-helices packed on one side. Similar folding patterns have been observed for other ribosomal proteins and may suggest an original RNA-interacting motif. Related topologies are also found in several other nucleic acid-interacting proteins and based on the assumption that the structure of the ribosome was established early in the molecular evolution, the possibility that an ancestral RNA-interacting motif in ribosomal proteins is the evolutionary origin for the nucleic acid-interacting domain in large classes of ribonucleic acid binding proteins should be considered.     

The Ribosome Can Prevent Aggregation of Partially Folded Protein Intermediates: Studies Using the Escherichia coli Ribosome

Background

Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone ‘foldases’ that are distinct from chaperone’ holdases’ that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC) located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA) is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains.

Results

We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII) and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein.

Conclusion

The ribosome can behave like a ‘holdase’ chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.     

棕色固氮菌突变种UW3部分提纯的固氮酶CrFe蛋白溶液中残存细菌铁蛋白的晶体生长及鉴定

从无钼、无氨而含铬的固氮培养基中生长的棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW3中纯化得到了部分纯的CrFe蛋白.在试图培养CrFe蛋白大晶体时发现,棕色晶体和砖红色晶体可同时或单独出现.SDS-PAGE和厌氧天然PAGE皆表明,棕色晶体主要由与固氮酶钼铁蛋白(Av1)类似大小的亚基(～60 kD)组成,而砖红色晶体则由～20kD亚基组成.免疫分析表明只有～60kD的亚基可与固氮酶钼铁蛋白的抗体反应,而～20kD亚基则无这种反应.在部分纯的CrFe蛋白溶液中,～20 kD的总蛋白含量远低于～60 kD蛋白的含量,表明由这种小亚基组成的蛋白只是CrFe蛋白溶液中的一种污染蛋白.用3,5-二氨基苯甲酸染色的天然电泳表明,形成砖红色和棕色晶体的蛋白是迁移率不同的两种含铁蛋白.质谱分析表明砖红色晶体蛋白为棕色固氮菌的细菌铁蛋白.分辨率为2.34 A的X射线衍射结果也表明,砖红色晶体属于H3空间群,晶胞参数为a=124.965A,b=124.965A和c=287.406 A.即将发表的三维结构解析表明,此砖红色晶体确为24聚体的细菌铁蛋白.