Stabilization of 70S Ribosomes by Spermidine

RIBOSOMAL subunits in Escherichia coli dissociate and reasso-ciate after each round of translation¹, but it is not known whether separation occurs at termination of protein synthesis², or whether they are released as “free” 70S ribosomes and subsequently dissociate³. The existence of free 70S ribosomes in vivo is controversial because sedimentation analyses of cell extracts using K⁺ as the monovalent cation demonstrate significant numbers of 70S ribosomes, but when Na⁺ is used 30S and 50S subunits predominate^4,5.     

羊驼皮肤中核糖体蛋白L34（RPL34）基因表达的分析

从羊驼皮肤eDNA文库中筛选到冗P￡34的克隆,经PCR鉴定并测序得知：一种克隆的大小为270bp（命名为尺P躬4,）,另一种克隆的大小为470bp（命名为RPL34Ⅱ）。序列分析发现,RPL34Ⅰ和Ⅱ在核苷酸水平上,5’UTR区不具有同源性,3’UTR区具有同源性;在氨基酸水平上,部分具有高度同源性;分子进化树分析表明二者具有不同的进化速率。此外,RPL34Ⅰ和Ⅱ对应于不同的转录本。通过以上结果分析,认为RPL34Ⅰ和RPL34Ⅱ二者同时在羊驼皮肤中表达,可能是RPIJ34的两个剪接体,发挥不同的生物学功能。     

Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation

Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process.     

Adaptive Potential of Wheat Ribosomes toward Heat Depends on the Large Ribosomal Subunit and Ribosomal Protein Phosphorylation

In a study of the translational efficiency of ribosomal subunits as a function of an in vivo temperature pretreatment, ribosomes were isolated from heat-pretreated (36°C) and reference (20°C) wheat seedlings (Triticum aestivum L.). The efficiency of recombined subunits in translating polyuridylic acid was assessed. A threefold increase in the rate of incorporation of phenylalanine by ribosomes from heat-pretreated plants was due to the large ribosomal subunit. This adaptive temperature effect was not correlated with a higher thermal stability of ribosomes or subunits from heat-pretreated seedlings, and two-dimensional gel electrophoresis failed to detect structural alterations of ribosomal proteins. Phosphorylation of ribosomal proteins in vitro showed no differences between ribosomes or subunits from heat-pretreated and reference plants. Incubation with [³²P]orthophosphate in vivo led to twice the amount of phosphate in ribosomal proteins from heat-pretreated wheat seedlings. This result is important with respect to the evaluation of the molecular basis of enhanced translational efficiency of ribosomes isolated from heat-pretreated wheat seedlings.     

The Synthesis of Ribosomes in E. coli: IV. The Synthesis of Ribosomal Protein and the Assembly of Ribosomes

The incorporation of C¹⁴ leucine into the protein moiety of ribosomes has been studied as a sequel to the studies of ribosomal RNA synthesis. In contrast to the latter studies, labeled leucine is incorporated directly into 50S and 30S ribosomes without measurable delay by precursor stages. There is, however, evidence of some transfer of radioactivity from the 43S group of particles to the 50S. The inhibition of protein synthesis by chloramphenicol results in the accumulation of material similar to the eosome—the primary precursor in ribosome synthesis. There is also evidence for the synthesis of some neosome. The results of the studies of ribosomal RNA and protein synthesis are combined into a model of ribosome synthesis. Finally, consideration is made of the significance of these studies of ribosome synthesis for general problems of protein synthesis and information transfer.     

Constitutive Production of 92-kDa Gelatinase B Can Be Suppressed by Alterations in Cell Shape

We have examined the effect that cell shape has on production of the 92-kDa gelatinase B, an enzyme of the matrix metalloproteinase family thought to contribute to the invasiveness of both normal and malignant cells. Using the agent poly(HEMA) and a human melanoma cell line that constitutively produces both the 72- and 92-kDa gelatinases, we have found that alteration in cell shape, that is, a change in cell "roundness," resulted in a specific loss of the constitutive production of the 92-kDa gelatinase B. To examine this phenomenon further, cells were treated with an inhibitor of actin polymerization, cytochalasin D. This treatment also resulted in a loss of 92-kDa gelatinase B production, provided the cells were treated with drug from the outset of the experiment. If the cells were allowed to attach and spread prior to drug exposure, no loss of 92-kDa gelatinase B production was observed. Similar to the poly (HEMA) results, cytochalasin D had little effect on production of the 72-kDa gelatinase A. Treatment with the tublin polymerization inhibitor colchicine had no effect on 92-kDa gelatinase B production, nor did growth of the cells as three-dimensional tumor spheroids, although an alteration in cell morphology was observed in both instances. This phenomenon was studied in another system, namely, HL-60 cells, which were induced to differentiate into macrophage-like cells in response to TPA treatment and consequently produce the 92-kDa gelatinase B. HL-60 cells treated with TPA and cytochalasin D failed to produce the 92-kDa gelatinase B. These results suggest that the 92-kDa gelatinase B can be regulated by alterations in cell shape but more specifically, by alterations in the organization of the actin cytoskeleton. Furthermore, the mechanism responsible for cell shape/actin cytoskeletal down-regulation of the 92-kDa gelatinase B may be common to many cell types competent to produce this enzymatic activity.     

Biases in Phylogenetic Estimation Can Be Caused by Random Sequence Segments

We consider the effects of fully or partially random sequences on the estimation of four-taxon phylogenies. Fully or partially random sequences occur when whole subsets of sequences or some sites for subsets of sequences are independent of sequence data for the other taxa. Random sequences can be a consequence of misalignment or because sites evolve at very fast rates in some portions of a tree, a situation that occurs especially in analyses involving deep divergence times. One might reasonably speculate that random sites will only add noise to the estimation of a phylogeny. We show that in the case that a random sequence is added to a three-taxa alignment, it is more likely to be a neighbor of the sequence corresponding to the longest branch in the three-taxon tree. Surprisingly, when only about half of the sites show randomness, a long-branch-repels form of small sample bias occurs, and when a minority of sites show randomness this becomes a long-branch-attraction bias again. The most serious bias, one that does not vanish with increasing sequence length, occurs when more than one sequence is partially random. If there is a large amount of overlap in the random sites for two sequences, those two sequences will be attracted to each other; otherwise, they will repel each other. Random sequences or sites can, therefore, cause complicated biases in phylogenetic inference. We suggest performing analyses with and without potentially saturated sequences and/or misaligned sites, to check that these biases are not affecting the inferred branching pattern.[Reviewing Editor: Dr. J. Rasmus Nielson]     

Perturbation of 60 S Ribosomal Biogenesis Results in Ribosomal Protein L5- and L11-dependent p53 Activation

Ribosomal proteins play an important role in p53 activation in response to nucleolar stress. Multiple ribosomal proteins, including L5, L11, L23, and S7, have been shown to bind to and inhibit MDM2, leading to p53 activation. However, it is not clear whether ribosomal protein regulation of MDM2 is specific to some, but not all ribosomal proteins. Here we show that L29 and L30, two ribosomal proteins from the 60 S ribosomal subunit, do not bind to MDM2 and do not inhibit MDM2-mediated p53 suppression, indicating that the ribosomal protein regulation of the MDM2-p53 feedback loop is specific. Interestingly, direct perturbation of the 60 S ribosomal biogenesis by knocking down either L29 or L30 drastically induced the level and activity of p53, leading to p53-depedent cell cycle arrest. This p53 activation was drastically inhibited by knockdown of L11 or L5. Consistently, knockdown of L29 or L30 enhanced the interaction of MDM2 with L11 and L5 and markedly inhibited MDM2-mediated p53 ubiquitination, suggesting that direct perturbation of 60 S ribosomal biogenesis activates p53 via L11- and L5-mediated MDM2 suppression. Mechanistically, knockdown of L30 or L29 significantly increased the NEDDylation and nuclear retention of L11. Knocking down endogenous NEDD8 suppressed p53 activation induced by knockdown of L30. These results demonstrate that NEDDylation of L11 plays a critical role in mediating p53 activation in response to perturbation of ribosomal biogenesis.     

Double-Stranded RNA-Activated Protein Kinase (PKR) Is Negatively Regulated by 60S Ribosomal Subunit Protein L18

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2α), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2α in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2α phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.     

Selectivity by Small-Molecule Inhibitors of Protein Interactions Can Be Driven by Protein Surface Fluctuations

Small-molecules that inhibit interactions between specific pairs of proteins have long represented a promising avenue for therapeutic intervention in a variety of settings. Structural studies have shown that in many cases, the inhibitor-bound protein adopts a conformation that is distinct from its unbound and its protein-bound conformations. This plasticity of the protein surface presents a major challenge in predicting which members of a protein family will be inhibited by a given ligand. Here, we use biased simulations of Bcl-2-family proteins to generate ensembles of low-energy conformations that contain surface pockets suitable for small molecule binding. We find that the resulting conformational ensembles include surface pockets that mimic those observed in inhibitor-bound crystal structures. Next, we find that the ensembles generated using different members of this protein family are overlapping but distinct, and that the activity of a given compound against a particular family member (ligand selectivity) can be predicted from whether the corresponding ensemble samples a complementary surface pocket. Finally, we find that each ensemble includes certain surface pockets that are not shared by any other family member: while no inhibitors have yet been identified to take advantage of these pockets, we expect that chemical scaffolds complementing these “distinct” pockets will prove highly selective for their targets. The opportunity to achieve target selectivity within a protein family by exploiting differences in surface fluctuations represents a new paradigm that may facilitate design of family-selective small-molecule inhibitors of protein-protein interactions.     

Phylogenetic Analysis of L4-Mediated Autogenous Control of the S10 Ribosomal Protein Operon

We investigated the regulation of the S10 ribosomal protein (r-protein) operon among members of the gamma subdivision of the proteobacteria, which includes Escherichia coli. In E. coli, this 11-gene operon is autogenously controlled by r-protein L4. This regulation requires specific determinants within the untranslated leader of the mRNA. Secondary structure analysis of the S10 leaders of five enterobacteria (Salmonella typhimurium, Citrobacter freundii, Yersinia enterocolitica, Serratia marcescens, and Morganella morganii) and two nonenteric members of the gamma subdivision (Haemophilus influenzae and Vibrio cholerae) shows that these foreign leaders share significant structural homology with the E. coli leader, particularly in the region which is critical for L4-mediated autogenous control in E. coli. Moreover, these heterologous leaders produce a regulatory response to L4 oversynthesis in E. coli. Our results suggest that an E. coli-like L4-mediated regulatory mechanism may operate in all of these species. However, the mechanism is not universally conserved among the gamma subdivision members, since at least one, Pseudomonas aeruginosa, does not contain the required S10 leader features, and its leader cannot provide the signals for regulation by L4 in E. coli. We speculate that L4-mediated autogenous control developed during the evolution of the gamma branch of proteobacteria.     

Mg2+ Dependence of 70 S Ribosomal Protein Flexibility Revealed by Hydrogen/Deuterium Exchange and Mass Spectrometry

The ribosome from Escherichia coli requires a specific concentration of Mg²⁺ to maintain the 70 S complex formation and allow protein synthesis, and then the structure must be stable and flexible. How does the ribosome acquire these conflicting factors at the same time? Here, we investigated the hydrogen/deuterium exchange of 52 proteins in the 70 S ribosome, which controlled stability and flexibility under various Mg²⁺ concentrations, using mass spectrometry. Many proteins exhibited a sigmoidal curve for Mg²⁺ concentration dependence, incorporating more deuterium at lower Mg²⁺ concentration. By comparing deuterium incorporation with assembly, we have discovered a typical mechanism of complexes for acquiring both stability and flexibility at the same time. In addition, we got information of the localization of flexibility in ribosomal function by the analysis of related proteins with stalk protein, tRNA, mRNA, and nascent peptide, and demonstrate the relationship between structure, assembly, flexibility, and function of the ribosome.     

Final Pre-40S Maturation Depends on the Functional Integrity of the 60S Subunit Ribosomal Protein L3

Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3′ end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles.     

A Role for Ribosomal Protein L5 in the Nuclear Import of 5S rRNA inXenopusOocytes

Prior to ribosome assembly, 5S ribosomal RNA (5S rRNA) binds to ribosomal protein L5 to form a stable ribonucleoprotein particle (5S RNP). We have analyzed the role of L5 binding in the nuclear targeting of 5S rRNA inXenopusoocytes, and have compared the nuclear import pathway of 5S RNPs with other karyophilic molecules. Nuclear import ofin vitro-generated 5S RNPs was found to be sensitive to three general inhibitors of nuclear pore complex-mediated translocation: ATP depletion, chilling, and wheat germ agglutinin. The initial rate and extent of net nuclear import was threefold greater with preassembled 5S RNPs than with 5S rRNA microinjected alone, suggesting that L5 binding is a prerequisite for nuclear accumulation. Nuclear import of 5S rRNA/5S RNPs is a facilitated process dependent on limiting factors, since nuclear import exhibited saturation kinetics. Not only was nuclear import of labeled 5S rRNA reduced in the presence of excess unlabeled 5S rRNA, but also in the presence of the synthetic karyophilic protein P(lys)-BSA. In contrast, import was not inhibited by U1 small nuclear RNA (snRNA) or U3 small nucleolar RNA (snoRNA). 5S rRNA/5S RNP nuclear import therefore appears to follow a pathway of molecular interactions similar to many karyophilic proteins, but not the methylguanosine cap-dependent U1 snRNA pathway or the cap-independent U3 snoRNA pathway.