Showing papers on "Ribosomal protein published in 2003"

Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway.

Abstract: The gene encoding p53 mediates a major tumor suppression pathway that is frequently altered in human cancers. p53 function is kept at a low level during normal cell growth and is activated in response to various cellular stresses. The MDM2 oncoprotein plays a key role in negatively regulating p53 activity by either direct repression of p53 transactivation activity in the nucleus or promotion of p53 degradation in the cytoplasm. DNA damage and oncogenic insults, the two best-characterized p53-dependent checkpoint pathways, both activate p53 through inhibition of MDM2. Here we report that the human homologue of MDM2, HDM2, binds to ribosomal protein L11. L11 binds a central region in HDM2 that is distinct from the ARF binding site. We show that the functional consequence of L11-HDM2 association, like that with ARF, results in the prevention of HDM2-mediated p53 ubiquitination and degradation, subsequently restoring p53-mediated transactivation, accumulating p21 protein levels, and inducing a p53-dependent cell cycle arrest by canceling the inhibitory function of HDM2. Interference with ribosomal biogenesis by a low concentration of actinomycin D is associated with an increased L11-HDM2 interaction and subsequent p53 stabilization. We suggest that L11 functions as a negative regulator of HDM2 and that there might exist in vivo an L11-HDM2-p53 pathway for monitoring ribosomal integrity.

The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes.

Abstract: Recent reports have increased our knowledge of the consecutive steps during 60S ribosome biogenesis substantially, but 40S subunit formation is less well understood. Here, we investigate the maturation of nucleolar 90S pre-ribosomes into cytoplasmic 40S pre-ribosomes. During the transition from 90S to 40S particles, the majority of non-ribosomal proteins (∼30 species) dissociate, and significantly fewer factors associate with 40S pre-ribosomes. Notably, some of these components are part of both early 90S and intermediate 40S pre-particles in the nucleolus (e.g. Enp1p, Dim1p and Rrp12p), whereas others (e.g. Rio2p and Nob1p) are found mainly on late cytoplasmic pre-40S subunits. Finally, temperature-sensitive mutants mapping either in earlier (enp1-1) or later (rio2-1) components exhibit defects in the formation and nuclear export of pre-40S subunits. Our data provide an initial biochemical map of the pre-40S ribosomal subunit on its path from the nucleolus to the cytoplasm. This pathway involves fewer changes in composition than seen during 60S biogenesis.

Comparative genomics of bacterial zinc regulons: Enhanced ion transport, pathogenesis, and rearrangement of ribosomal proteins

Abstract: Zinc is an important component of many proteins, but in large concentrations it is poisonous to the cell. Thus its transport is regulated by zinc repressors ZUR of proteobacteria and Gram-positive bacteria from the Bacillus group and AdcR of bacteria from the Streptococcus group. Comparative computational analysis allowed us to identify binding signals of ZUR repressors GAAATGTTATANTATAACATTTC for γ-proteobacteria, GTAATGTAATAACATTAC for the Agrobacterium group, GATATGTTATAACATATC for the Rhododoccus group, TAAATCGTAATNATTACGATTTA for Gram-positive bacteria, and TTAACYRGTTAA of the streptococcal AdcR repressor. In addition to known transporters and their paralogs, zinc regulons were predicted to contain a candidate component of the ATP binding cassette, zinT (b1995 in Escherichia coli and yrpE in Bacillus subtilis). Candidate AdcR-binding sites were identified upstream of genes encoding pneumococcal histidine triad (PHT) proteins from a number of pathogenic streptococci. Protein functional analysis of this family suggests that PHT proteins are involved in the invasion process. Finally, repression by zinc was predicted for genes encoding a variety of paralogs of ribosomal proteins. The original copies of all these proteins contain zinc-ribbon motifs and thus likely bind zinc, whereas these motifs are destroyed in zinc-regulated paralogs. We suggest that the induction of these paralogs in conditions of zinc starvation leads to their incorporation in a fraction of ribosomes instead of the original ribosomal proteins; the latter are then degraded with subsequent release of some zinc for the utilization by other proteins. Thus we predict a mechanism for maintaining zinc availability for essential enzymes.

The DEAD-box RNA helicase SrmB is involved in the assembly of 50S ribosomal subunits in Escherichia coli.

Abstract: Summary Ribosome assembly in Escherichia coli involves 54 ribosomal proteins and three RNAs. Whereas functional subunits can be reconstituted in vitro from the isolated components, this process requires long incubation times and high temperatures compared with the in vivo situation, suggesting that non-ribosomal factors facilitate assembly in vivo. Here, we show that SrmB, a putative DEAD-box RNA helicase, is involved in ribosome assembly. The deletion of the srmB gene causes a slow-growth phenotype at low temperature. Polysome profile analyses of the corresponding cells reveal a deficit in free 50S ribosomal subunits and the accumulation of a new particle sedimenting around 40S. Analysis of the ribosomal RNA and protein contents of the 40S particle indicates that it represents a large subunit that is incompletely assembled. In particular, it lacks L13, one of the five ribosomal proteins that are essential for the early assembly step in vitro. Sucrose gradient fractionation also shows that, in wild-type cells, SrmB associates with a pre50S particle. From our results, we propose that SrmB is involved in an early step of 50S assembly that is necessary for the binding of L13. This step may consist of a structural rearrangement that, at low temperature, cannot occur without the assistance of this putative RNA helicase.

Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea

Abstract: Small nucleolar RNAs (designated as snoRNAs in Eukarya or sRNAs in Archaea) can be grouped into H/ACA or C/D box snoRNA (sRNA) subclasses. In Eukarya, H/ACA snoRNAs assemble into a ribonucleoprotein (RNP) complex comprising four proteins: Cbf5p, Gar1p, Nop10p and Nhp2p. A homolog for the Nhp2p protein has not been identified within archaeal H/ACA RNPs thus far, while potential orthologs have been identified for the other three proteins. Nhp2p is related, particularly in the middle portion of the protein sequence, to the archaeal ribosomal protein and C/D box protein L7Ae. This finding suggests that L7Ae may be able to substitute for the Nhp2p protein in archaeal H/ACA sRNAs. By band shift assays, we have analyzed in vitro the interaction between H/ACA box sRNAs and protein L7Ae from the archaeon Archaeoglobus fulgidus. We present evidence that L7Ae forms specific complexes with three different H/ACA sRNAs, designated as Afu-4, Afu-46 and Afu-190 with an apparent Kd ranging from 28 to 100 nM. By chemical and enzymatic probing we show that distinct bases located within bulges or loops of H/ACA sRNAs interact with the L7Ae protein. These findings are corroborated by mutational analysis of the L7Ae binding site. Thereby, the RNA motif required for L7Ae binding exhibits a structure, designated as the K-turn, which is present in all C/D box sRNAs. We also identified four H/ACA RNAs from the archaeal species Pyrococcus which exhibit the K-turn motif at a similar position in their structure. These findings suggest a triple role for L7Ae protein in Archaea, e.g. in ribosomes as well as H/ACA and C/D box sRNP biogenesis and function by binding to the K-turn motif.

RNA as a Drug Target: The Case of Aminoglycosides

Abstract: These structures definitively show thatantibioticspredominantly target ribosomal RNA molecules,rather than ribosomal proteins. Each crystal structure of thesame particle bound to various antibiotics, partners, andcofactorsimplicatedintheproteinsynthesisprovidesasnapshotof the inhibitory process at the molecular level. When assem-bled, these individual pictures help to reconstruct the wholecourseofactionandindirectlyimproveourunderstandingoftheprotein synthesis process. Crystal structures have also beensolved for an isolated domain of the ribosome in complex withseveral antibiotics from the aminoglycoside family.

Internal telomeric repeats and 'TCP domain' protein-binding sites co-operate to regulate gene expression in Arabidopsis thaliana cycling cells

Abstract: We have focused our interest on two cis-regulatory elements, named site II motif and telo box, identified within the promoter of plant proliferating cellular nuclear antigen (PCNA) and putatively involved in meristematic expression of the gene. A conserved topological association between site II motifs and telo boxes is observed in the promoter of numerous genes expressed in cycling cells, including several cell cycle-related genes and 153 Arabidopsis genes encoding ribosomal proteins. Meristematic expression of a GUS reporter gene was observed in plants under the control of Arabidopsis site II motif within a minimal promoter. This expression is strongly enhanced by addition of a telo box within this chimaeric promoter. We showed by gel retardation experiments that the site II motif is a target for several DNA-binding activities present in Arabidopsis crude cell extract and can bind a transcription factor, At-TCP20, from the Teosinte branched 1, Cycloidea, PCF (TCP)-domain protein family. In yeast two-hybrid experiments, At-TCP20 appears to be a potential partner of AtPuralpha, which was previously shown to bind telo boxes. An important consequence of this analysis is to reveal new and conserved regulatory processes concerning the regulation of plant ribosomal gene expression in cycling cells. The implication of these observations in plant-specific developmental pathways is discussed.

The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome

Abstract: The signal recognition particle (SRP) from Escherichia coli, composed of Ffh protein and 4.5S RNA, mediates membrane targeting of translating ribosomes displaying a signal or signal-anchor sequence. SRP binds at the peptide exit of the large ribosomal subunit. Structural details of the interaction are not known. Here, the position of Ffh or SRP on the ribosome was probed by using site-specific UV-induced crosslinking by p-azidophenacyl bromide (AzP) attached to a number of cysteine residues engineered into surface positions of Ffh. Efficient crosslinking to vacant ribosomes took place from two positions (AzP17 and AzP25) in the N domain of Ffh, both with Ffh and SRP. Both AzP17 and AzP25 were predominantly crosslinked to ribosomal protein L23 that is located at the peptide exit of the 50S subunit. The SRP receptor, FtsY, did not change the crosslink pattern, whereas the presence of a nascent signal peptide on the ribosome resulted in a second crosslink between Ffh(AzP17) and protein L23, indicating that binding to the nascent signal peptide induced a slightly different arrangement of SRP on the ribosome. These results indicate a model of the topographical arrangement of SRP at the peptide exit of the 50S ribosomal subunit.

Properties of human mitochondrial ribosomes.

Abstract: Mammalian mitochondrial ribosomes (55S) differ unexpectedly from bacterial (70S) and cytoplasmic ribosomes (80S), as well as other kinds of mitochondrial ribosomes. Typical of mammalian mitochondrial ribosomes, the bovine mitochondrial ribosome has been developed as a model system for the study of human mitochondrial ribosomes, to address several questions related to the structure, function, biosynthesis and evolution of these interesting ribosomes. Bovine mitochondrial ribosomal proteins (MRPs) from each subunit have been identified and characterized with respect to individuality and electrophoretic properties, amino acid sequence, topographic disposition, RNA binding properties, evolutionary relationships and reaction with affinity probes of ribosomal functional domains. Several distinctive properties of these ribosomes are being elucidated, including their antibiotic susceptibility and composition. Human mitochondrial ribosomes lack several of the major RNA stem structures of bacterial ribosomes but they contain a correspondingly higher protein content (as many as 80 proteins), suggesting a model where proteins have replaced RNA structural elements during the evolution of these ribosomes. Despite their lower RNA content they are physically larger than bacterial ribosomes, because of the 'extra' proteins they contain. The extra proteins in mitochondrial ribosomes are 'new' in the sense that they are not homologous to proteins in bacterial or cytoplasmic ribosomes. Some of the new proteins appear to be bifunctional. All of the mammalian MRPs are encoded in nuclear genes (a separate set from those encoding cytoplasmic ribosomal proteins) which are evolving more rapidly than those encoding cytoplasmic ribosomal proteins. The MRPs are imported into mitochondria where they assemble coordinately with mitochondrially transcribed rRNAs into ribosomes that are responsible for translating the 13 mRNAs for essential proteins of the oxidative phosphorylation system.

Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

Abstract: As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro–synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.

The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively.

Abstract: We characterized two essential putative GTPases, Nog1p and Lsg1p, that are found associated with free 60S ribosomal subunits affinity purified with the nuclear export adapter Nmd3p. Nog1p and Lsg1p are nucleolar and cytoplasmic, respectively, and are not simultaneously on the same particle, reflecting the path of Nmd3p shuttling in and out of the nucleus. Conditional mutants of both NOG1 and LSG1 are defective in 60S subunit biogenesis and display diminished levels of 60S subunits at restrictive temperature. Mutants of both genes also accumulate the 60S ribosomal reporter Rpl25-eGFP in the nucleolus, suggesting that both proteins are needed for subunit export from the nucleolus. Since Lsg1p is cytoplasmic, its role in nuclear export is likely to be indirect. We suggest that Lsg1p is needed to recycle an export factor(s) that shuttles from the nucleus associated with the nascent 60S subunit.

A Salvage Pathway for Protein Synthesis: tmRNA and Trans-Translation

Abstract: ▪ Abstract Transfer-messenger RNA (tmRNA, or SsrA), found in all eubacteria, has both transfer and messenger RNA activity. Relieving ribosome stalling by a process called trans-translation, tmRNAala enters the ribosome and adds its aminoacylated alanine to the nascent polypeptide. The original mRNA is released and tmRNA becomes the template for translation of a 10-amino-acid tag that signals for proteolytic degradation. Although essential in a few bacterial species, tmRNA is nonessential in Escherichia coli and many other bacteria. Proteins known to be associated with tmRNA include SmpB, ribosomal protein S1, RNase R, and phosphoribosyl pyrophosphate. SmpB, having no other known function, is essential for tmRNA activity. trans-translation operates within ribosomes stalled both at the end of truncated mRNAs and at rare codons and some natural termination sites. Both the release of stalled ribosomes and the subsequent degradation of tagged proteins are important consequences of trans-translation.

Assembly of the 30S ribosomal subunit.

Abstract: Ribosomes are large macromolecular complexes responsible for cellular protein synthesis. The smallest known cytoplasmic ribosome is found in prokaryotic cells; these ribosomes are about 2.5 MDa and contain more than 4000 nucleotides of RNA and greater than 50 proteins. These components are distributed into two asymmetric subunits. Recent advances in structural studies of ribosomes and ribosomal subunits have revealed intimate details of the interactions within fully assembled particles. In contrast, many details of how these massive ribonucleoprotein complexes assemble remain elusive. The goal of this review is to discuss some crucial aspects of 30S ribosomal subunit assembly.

Late cytoplasmic maturation of the small ribosomal subunit requires RIO proteins in Saccharomyces cerevisiae.

Abstract: Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation. We have recently demonstrated that Rrp10p/Rio1p is such a factor. By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p. Rio2p homologues were identified throughout the Archaea and metazoan species. We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation. In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature. Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles. These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.

Transcriptional Regulation and Signature Patterns Revealed by Microarray Analyses of Streptococcus pneumoniae R6 Challenged with Sublethal Concentrations of Translation Inhibitors

Abstract: The effects of sublethal concentrations of four different classes of translation inhibitors (puromycin, tetracycline, chloramphenicol, and erythromycin) on global transcription patterns of Streptococcus pneumoniae R6 were determined by microarray analyses. Consistent with the general mode of action of these inhibitors, relative transcript levels of genes that encode ribosomal proteins and translation factors or that mediate tRNA charging and amino acid biosynthesis increased or decreased, respectively. Transcription of the heat shock regulon was induced only by puromycin or streptomycin treatment, which lead to truncation or mistranslation, respectively, but not by other antibiotics that block translation, transcription, or amino acid charging of tRNA. In contrast, relative transcript amounts of certain genes involved in transport, cellular processes, energy metabolism, and purine nucleotide ( pur ) biosynthesis were changed by different translation inhibitors. In particular, transcript amounts from a pur gene cluster and from purine uptake and salvage genes were significantly elevated by several translation inhibitors, but not by antibiotics that target other cellular processes. Northern blotting confirmed increased transcript amounts from part of the pur gene cluster in cells challenged by translation inhibitors and revealed the presence of a 10-kb transcript. Purine metabolism genes were negatively regulated by a homologue of the PurR regulatory protein, and full derepression in a Δ purR mutant depended on optimal translation. Unexpectedly, hierarchical clustering of the microarray data distinguished among the global transcription patterns caused by antibiotics that inhibit different steps in the translation cycle. Together, these results show that there is extensive control of transcript amounts by translation in S. pneumoniae , especially for de novo purine nucleotide biosynthesis. In addition, these global transcription patterns form a signature that can be used to classify the mode of action and potential mechanism of new translation inhibitors.

Heat‐stress‐dependency and developmental modulation of gene expression: the potential of house‐keeping genes as internal standards in mRNA expression profiling using real‐time RT‐PCR

Abstract: The potential of different house-keeping genes for their use as internal standards of gene expression under changing environmental conditions and in different organs of plants was assessed. Using real-time PCR mRNA levels were precisely quantified for preselected actin and ribosomal protein genes in Arabidopsis thaliana (L.) Heinh. and Nicotiana tabacum L. grown at normal temperature and following heat stress. In tobacco leaves the mRNA levels of the constitutively expressed ribosomal protein gene Nt-L25 and the actin genes Nt-ACT9 and At-ACT66 were strongly reduced (to approximately 10%) during heat stress. Heat stress applied at the temperature optimum (37 degrees C) for elicitation of a heat stress response to Arabidopsis leaves resulted in a strong induction (several thousand-fold) of the mRNA heat shock protein genes, At-HSP17.6 and At-HSP18.2. Concomitantly, the mRNA levels of constitutively expressed actin 2 (At-ACT2) and ribosomal protein L23 (At-L23a) genes were reduced to approximately 50% of the levels in leaves incubated at room temperature. Conversely, under severe heat stress conditions (44 degrees C), the induction of At-HSP17.6 and At-HSP18.2 mRNAs was insignificant, the mRNA levels of At-ACT2 remained at approximately the same levels as in leaves incubated at room temperature, whereas the mRNA level of At-L23 declined. The mRNA levels of At-ACT2 and At-L23a examined in stem, flower and siliques of Arabidopsis plants grown under non-stress condition showed differential alterations; the mRNA level of ribosomal protein L23 correlates with the metabolic activity of tissues. The potential use of house-keeping gene expression as standards in expression profiling and the mechanisms modulating the mRNA levels are discussed.

Interaction of Escherichia coli RNA polymerase with the ribosomal protein S1 and the Sm-like ATPase Hfq.

Abstract: We report evidence that ribosomal protein S1 and nucleic acid-binding protein Hfq copurify in molar ratios with RNA polymerase (RNAP). Purified S1 associates independently with RNAP, and Hfq binding to polymerase occurs in the presence of S1. Looking for a functional role of the RNAP-S1-Hfq association, we studied the effects of S1 and Hfq on transcription and coupled transcription-translation. S1 was capable of significant stimulation of the RNAP transcriptional activity from a number of promoters; the stimulatory effect was observed on linear as well as supercoiled DNA templates. In addition, we present biochemical and genetic evidence of ATPase activity associated with the Sm-like hexameric nucleic acid-binding protein Hfq. The limited sequence homology between Hfq and known ATP-utilizing enzymes suggests a new class of ATPases.

Microorganism Identification by Matrix-Assisted Laser/Desorption Ionization Mass Spectrometry and Model-Derived Ribosomal Protein Biomarkers

Abstract: An improved data analysis method is described for rapid identification of intact microorganisms from MALDI-TOF-MS data. The method makes no use of mass spectral fingerprints. Instead, a microorganism database is automatically generated that contains biomarker masses derived from ribosomal protein sequences and a model of N-terminal Met loss. We quantitatively validate the method via a blind study that seeks to identify microorganisms with known ribosomal protein sequences. We also include in the database microorganisms with incompletely known sets of ribosomal proteins to test the specificity of the method. With an optimal MALDI protocol, and at the 95% confidence level, microorganisms represented in the database with 20 or more biomarkers (i.e., those with complete or nearly completely sequenced genomes) are correctly identified from their spectra 100% of the time, with no incorrect identifications. Microorganisms with seven or less biomarkers (i.e., incompletely sequenced genomes) are either not identif...

A comparison of three fission yeast mitochondrial genomes.

Abstract: The fission yeasts are members of the fungal order Schizosaccharomycetales, a candidate deep-diverging group within Ascomycota. Although a great deal of molecular information is available from Schizosaccharomyces pombe, a model eukaryote, very little is available from other members of this group. In order to better characterize mitochondrial genome evolution in this fungal lineage, the mitochondrial DNA (mtDNA) of two additional fission yeasts, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus var. japonicus, was sequenced. Whereas the mtDNA of S.pombe is only 19 431 bp, the mtDNA of S.octosporus is 44 227 bp, and that of S.japonicus var. japonicus is over 80 kb. The size variation of these mtDNAs is due largely to non-coding regions. The gene content in the latter two mtDNAs is almost identical to that of the completely sequenced S.pombe mtDNA, which encodes 25 tRNA species, the large and small mitochondrial ribosomal RNAs (rnl and rns), the RNA component of mitochondrial RNaseP (rnpB), mitochondrial small subunit ribosomal protein 3 (rps3), cytochrome oxidase subunits 1, 2 and 3 (cox1, cox2 and cox3) and ATP-synthase subunits 6, 8 and 9 (atp6, atp8 and atp9). However, trnI2(cau) (C modified to lysidine) is absent in the S.octosporus mtDNA, as are corresponding ATA codons in its protein-coding genes, and rps3 and rnpB are not found in the mtDNA of S.japonicus var. japonicus. The mtDNA of S.octosporus contains five double hairpin elements, the first report of these elements in an ascomycete. This study provides further evidence in favor of the mobility of these elements, and supports their role in mitochondrial genome rearrangement. The results of our phylogenetic analysis support the monophyly of the Schizosaccharomycetales, but question their grouping within the Archiascomycota.

Proteomic identification of all plastid‐specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit

Abstract: Six ribosomal proteins are specific to higher plant chloroplast ribosomes [Subramanian, A.R. (1993) Trends Biochem. Sci.18, 177–180]. Three of them have been fully characterized [Yamaguchi, K., von Knoblauch, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455–28465; Yamaguchi, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28466–28482]. The remaining three plastid-specific ribosomal proteins (PSRPs), all on the small subunit, have now been characterized (2D PAGE, HPLC, N-terminal/internal peptide sequencing, electrospray ionization MS, cloning/ sequencing of precursor cDNAs). PSRP-3 exists in two forms (α/β, N-terminus free and blocked by post-translational modification), whereas PSRP-2 and PSRP-4 appear, from MS data, to be unmodified. PSRP-2 contains two RNA-binding domains which occur in mRNA processing/stabilizing proteins (e.g. U1A snRNP, poly(A)-binding proteins), suggesting a possible role for it in the recruiting of stored chloroplast mRNAs for active protein synthesis. PSRP-3 is the higher plant orthologue of a hypothetical protein (ycf65 gene product), first reported in the chloroplast genome of a red alga. The ycf65 gene is absent from the chloroplast genomes of higher plants. Therefore, we suggest that Psrp-3/ycf65, encoding an evolutionarily conserved chloroplast ribosomal protein, represents an example of organelle-to-nucleus gene transfer in chloroplast evolution. PSRP-4 shows strong homology with Thx, a small basic ribosomal protein of Thermus thermophilus 30S subunit (with a specific structural role in the subunit crystallographic structure), but its orthologues are absent from Escherichia coli and the photosynthetic bacterium Synechocystis. We would therefore suggest that PSRP-4 is an example of gene capture (via horizontal gene transfer) during chloro-ribosome emergence. Orthologues of all six PSRPs are identifiable in the complete genome sequence of Arabidopsis thaliana and in the higher plant expressed sequence tag database. All six PSRPs are nucleus-encoded. The cytosolic precursors of PSRP-2, PSRP-3, and PSRP-4 have average targeting peptides (62, 58, and 54 residues long), and the mature proteins are of 196, 121, and 47 residues length (molar masses, 21.7, 13.8 and 5.2 kDa), respectively. Functions of the PSRPs as active participants in translational regulation, the key feature of chloroplast protein synthesis, are discussed and a model is proposed.

Regulated Phosphorylation of 40S Ribosomal Protein S6 in Root Tips of Maize

Abstract: Ribosomal protein S6 (RPS6) is located in the mRNA binding site of the 40S subunit of cytosolic ribosomes. Two maize (Zea mays) rps6 genes were identified that encode polypeptides (30 kD, 11.4 pI) with strong primary amino acid sequence and predicted secondary structure similarity to RPS6 of other eukaryotes. Maize RPS6 was analyzed by the use of two-dimensional gel electrophoresis systems, in vivo labeling with [(32)P]P(i) and immunological detection. Nine RPS6 isoforms were resolved in a two-dimensional basic-urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry performed on trypsin-digested isoforms identified four serine (Ser) and one threonine (Thr) residue in the carboxy-terminal region as phosphorylation sites (RRS(238)KLS(241)AAAKAS(247)AAT(250)S(251)A-COOH). Heterogeneity in RPS6 phosphorylation was a consequence of the presence of zero to five phosphorylated residues. Phosphorylated isoforms fell into two groups characterized by (a) sequential phosphorylation of Ser-238 and Ser-241 and (b) the absence of phospho-Ser-238 and presence of phospho-Ser-241. The accumulation of hyper-phosphorylated isoforms with phospho-Ser-238 was reduced in response to oxygen deprivation and heat shock, whereas accumulation of these isoforms was elevated by cold stress. Salt and osmotic stress had no reproducible effect on RPS6 phosphorylation. The reduction in hyper-phosphorylated isoforms under oxygen deprivation was blocked by okadaic acid, a Ser/Thr phosphatase inhibitor. By contrast, the recovery of hyper-phosphorylated isoforms upon re-oxygenation was blocked by LY-294002, an inhibitor of phosphatidylinositol 3-kinases. Thus, differential activity of phosphatase(s) and kinase(s) determine complex heterogeneity in RPS6 phosphorylation.

X-ray crystal structures of the WT and a hyper-accurate ribosome from Escherichia coli

Abstract: Protein biosynthesis on the ribosome requires accurate reading of the genetic code in mRNA. Two conformational rearrangements in the small ribosomal subunit, a closing of the head and body around the incoming tRNA and an RNA helical switch near the mRNA decoding site, have been proposed to select for complementary base-pairing between mRNA codons and tRNA anticodons. We determined x-ray crystal structures of the WT and a hyper-accurate variant of the Escherichia coli ribosome at resolutions of 10 and 9 A, respectively, revealing that formation of the intact 70S ribosome from its two subunits closes the conformation of the head of the small subunit independent of mRNA decoding. Moreover, no change in the conformation of the switch helix is observed in two steps of tRNA discrimination. These 70S ribosome structures indicate that mRNA decoding is coupled primarily to movement of the small subunit body, consistent with previous proposals, whereas closing of the head and the helical switch may function in other steps of protein synthesis.