Semliki Forest virus capsid protein associates with the 60S ribosomal subunit in infected cells.

Semlike forest virus capsid protein cosedimented with the large ribosomal subunit at 60S in sucrose gradients after treatment of cytoplasm from infected cells with Triton X-100 and EDTA. In CsCl gradients the capsid protein banded with the subunit at a density of 1.56 to 1.57 g/cm3. Most of the capsid protein could be detached from the 60S structure by treatment with 0.8 M KCl. The ribonucleoprotein of the 26S RNA had a sedimentation value of 53S and a density of 1.50 g/cm3 and could thus be separated from the 60S structure. The data suggest that the capsid protein binds to the large ribosomal subunit, but not to the viral 26S RNA.     

Ribosomal history reveals origins of modern protein synthesis

The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world.     

Ribosomal RNA synthesis in mitochondria of Neurospora crassa

Ribosomal RNA synthesis in Neurospora crassa mitochondria has been investigated by continuous labeling with [5-³H]uracil and pulse-chase experiments. A short-lived 32 S mitochondrial RNA was detected, along with two other short-lived components; one slightly larger than large subunit ribosomal RNA, and the other slightly larger than small subunit ribosomal RNA. The experiments give support to the possibility that 32 S RNA is the precursor of large and small subunit ribosomal RNA's. Both mature ribosomal RNA's compete with 32 S RNA in hybridization to mitochondrial DNA. Quantitative results from such hybridization-competition experiments along with measurements of electrophoretic mobility have been used to construct a molecular size model for synthesis of mitochondrial ribosomal RNA's. The large molecular weight precursor (32 S) of both ribosomal RNA's appears to be 2.4 × 10⁶ daltons in size. Maturation to large subunit RNA (1.28 × 10⁶ daltons) is assumed to involve an intermediate ~1.6 × 10⁶ daltons in size, while cleavage to form small subunit RNA (0.72 × 10⁶ daltons) presumably involves a 0.9 × 10⁶ dalton intermediate. In the maturation process ~22% of the precursor molecule is lost. As is the case for ribosomal RNA's, the mitochondrial precursor RNA has a strikingly low G + C content.     

Transient association of Semliki Forest virus capsid protein with ribosomes.

HeLa cells infected with Semliki Forest virus were exposed to [35S]methionine for 1 min and chased for various periods. The analysis of labeled ribonucleoproteins showed that the viral capsid protein associated first with the large ribosomal subunit in polysomes, from which it was chased to assembling nucleocapsids and to free monosomes.     

The binding of ribosomal protein S4 does not change the gross conformation of the 16 S RNA.

The binding of ribosomal protein S4 to the 16 S RNA does not result in a large shape or conformational change in the 16 S RNA under the conditions of reconstitution. The sedimentation coefficient, frictional coefficient ratio, and effective hydrodynamic radius of the 16 S RNA.protein S4 complex are very similar to those obtained for the 16 S RNA free in solution. Only subtle conformational differences were obtained in the comparison of the complex and free 16 S RNA by circular dichroism. Thus, extensive organization of the 16 S RNA by ribosomal protein S4 is not a step in the process of self-assembly of the 30 S subunit.     

Structure of single-stranded nucleic acids in the presence of ribosomal protein S1.

We have developed a new method for mounting nucleic acids and nucleic acidprotein complexes for high-resolution electron microscopy, and have used it to characterize the interaction between ribosomal protein S1 and single-stranded nucleic acids. We find that SI unwinds most, but not all of the secondary structure present in MS2 RNA and øX174 viral DNA. The binding of S1 to DNA and RNA is not highly co-operative, and has a stoichiometry of one S1 per 10 to 15 nucleotides. We have not observed any tendency for S1 nucleic acid complexes to form aggregates in either 0·01 m-Na⁺ or 0·1 m-Na⁺. An analogous protein isolated from the 30 S ribosomal subunit of Caulobacter crescentus is indistinguishable from Escherichia coli S1 in these studies. The mono-N-ethylmaleimide derivative of E. coli S1 will bind to both MS2 RNA and øX174 viral DNA with a stoichiometry of one N-ethylmaleimide-S1 per 10 to 15 nucleotides, but will not unwind the secondary structure of either of them.     

Alteration of ribosomal protein S2 in kasugamycin-resistant mutant derived from Escherichia coli AB312

A new type of kasugamycin-resistant mutant has been isolated from $\text{E. coli}$ K12, strain AB312 (Hfr, $\text{lac,thr,leu,thi,strA,fus}$). In a cell-free protein-synthetic system, the resistance is localized in the ribosome but not in the supernatant fraction. On initiation complex formation, the resistance is associated with the washed ribosome but not with initiation factors. In reconstitution of the 30S ribosomal subunit, the resistance is due to the protein(s) but not to 16S RNA. In two-dimensional electrophoresis, protein S2 is deficient in the 30S ribosomal subunit of kasugamycin-resistant mutant. The results indicate that the kasugamycin-resistance is attributed to alteration of ribosomal protein S2.     

Structure and function in the herpes simplex virus 1 RNA-binding protein U(s)11: mapping of the domain required for ribosomal and nucleolar association and RNA binding in vitro.

The herpes simplex virus 1 US11 protein is an RNA-binding regulatory protein that specifically and stably associates with 60S ribosomal subunits and nucleoli and is incorporated into virions. We report that US11/ beta-galactosidase fusion protein expressed in bacteria bound to rRNA from the 60S subunit and not the 40S subunit. This binding reflects the specificity of ribosomal subunit association. Analyses of deletion mutants of the US11 gene showed that specific RNA binding activity, nucleolar localization, and association with 60S ribosomal subunits were found to map to the amino acid sequences of the carboxyl terminus of US11 protein, suggesting that these activities all reflect specific binding of US11 to large subunit rRNA. The carboxyl-terminal half of the protein consists of a regular tripeptide repeat of the sequence RXP and constitutes a completely novel RNA-binding domain. All of the mutant US11 proteins could be incorporated into virus particles, suggesting that the signal for virion incorporation either is at the amino-terminal four amino acids or is redundant in the protein.     

Mitochondrial ribosomal RNA genes of yeast: Their mutations and a common nuclear suppressor

Summary Due to the absence of repetition of the rRNA genes in S. cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system. We describe here a novel thermosensitive mutation, ts1297, localized by rho^- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene. Defective mutations of the small rRNA have not been reported so far.In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced. The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal. However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca²⁺ in place of Mg²⁺. The 50S particles seem to be completely degraded under normal conditions of extraction with Mg²⁺.The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU₁₀₁. The SU₁₀₁ mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene.These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits.     

Assembly of the 30S ribosomal subunit: positioning ribosomal protein S13 in the S7 assembly branch

Studies of Escherichia coli 30S ribosomal subunit assembly have revealed a hierarchical and cooperative association of ribosomal proteins with 16S ribosomal RNA; these results have been used to compile an in vitro 30S subunit assembly map. In single protein addition and omission studies, ribosomal protein S13 was shown to be dependent on the prior association of ribosomal protein S20 for binding to the ribonucleoprotein particle. While the overwhelming majority of interactions revealed in the assembly map are consistent with additional data, the dependency of S13 on S20 is not. Structural studies position S13 in the head of the 30S subunit > 100 A away from S20, which resides near the bottom of the body of the 30S subunit. All of the proteins that reside in the head of the 30S subunit, except S13, have been shown to be part of the S7 assembly branch, that is, they all depend on S7 for association with the assembling 30S subunit. Given these observations, the assembly requirements for S13 were investigated using base-specific chemical footprinting and primer extension analysis. These studies reveal that S13 can bind to 16S rRNA in the presence of S7, but not S20. Additionally, interaction between S13 and other members of the S7 assembly branch have been observed. These results link S13 to the 3' major domain family of proteins, and the S7 assembly branch, placing S13 in a new location in the 30S subunit assembly map where its position is in accordance with much biochemical and structural data.     

The RNA-binding domain of ribosomal protein L11 recognizes an rRNA tertiary structure stabilized by both thiostrepton and magnesium ion

Antibiotics that inhibit ribosomal function may do so by one of several mechanisms, including the induction of incorrect RNA folding or prevention of protein and/or RNA conformational transitions. Thiostrepton, which binds to the ‘GTPase center’ of the large subunit, has been postulated to prevent conformational changes in either the L11 protein or rRNA to which it binds. Scintillation proximity assays designed to look at the binding of the L11 C-terminal RNA-binding domain to a 23S ribosomal RNA (rRNA) fragment, as well as the ability of thiostrepton to induce that binding, were used to demonstrate the role of Mg²⁺, L11 and thiostrepton in the formation and maintenance of the rRNA fragment tertiary structure. Experiments using these assays with both an Escherichia coli rRNA fragment and a thermostable variant of that RNA show that Mg²⁺, L11 and thiostrepton all induce the RNA to fold to an essentially identical tertiary structure.     

Functional interaction between RNA helicase II/Gu(alpha) and ribosomal protein L4

RNA helicase II/Gu(alpha) is a multifunctional nucleolar protein involved in ribosomal RNA processing in Xenopus laevis oocytes and mammalian cells. Downregulation of Gu(alpha) using small interfering RNA (siRNA) in HeLa cells resulted in 80% inhibition of both 18S and 28S rRNA production. The mechanisms underlying this effect remain unclear. Here we show that in mammalian cells, Gu(alpha) physically interacts with ribosomal protein L4 (RPL4), a component of 60S ribosome large subunit. The ATPase activity of Gu(alpha) is important for this interaction and is also necessary for the function of Gu(alpha) in the production of both 18S and 28S rRNAs. Knocking down RPL4 expression using siRNA in mouse LAP3 cells inhibits the production of 47/45S, 32S, 28S, and 18S rRNAs. This inhibition is reversed by exogenous expression of wild-type human RPL4 protein but not the mutant form lacking Gu(alpha)-interacting motif. These observations have suggested that the function of Gu(alpha) in rRNA processing is at least partially dependent on its ability to interact with RPL4.     

Identification of a U8 snoRNA-specific binding protein

Eukaryotic nucleoli contain a large and diverse population of small nucleolar ribonucleoprotein particles (snoRNPs) that play diverse and essential roles in ribosome biogenesis. We previously demonstrated that U8 snoRNP is essential for processing of both 5.8 and 28 S rRNA. The RNA component of the U8 RNP particle is necessary but not sufficient for processing. Using an electrophoretic mobility sift assay, we enriched for U8-specific binding proteins from Xenopus ovary extracts. UV cross-linking reactions with partially purified fractions implicated a 29-kDa protein directly binding to U8 RNA. This protein interacted specifically and with high affinity with U8 snoRNA; it did not bind other snoRNAs and is probably not a common component of all snoRNPs. This is the first report of a protein component specific to an snoRNP essential for processing of the large ribosomal subunit in vertebrates.     

The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes.

Throughout the purification of the mdm-2 or mdm-2-p53 protein complexes, a protein with a molecular weight of 34,000 was observed to copurify with these proteins. Several monoclonal antibodies directed against distinct epitopes in the mdm-2 or p53 protein coimmunoprecipitated this 34,000-molecular-weight protein, which did not react to p53 or mdm-2 polyclonal antisera in a Western immunoblot. The N-terminal amino acid sequence of this 34,000-molecular-weight protein demonstrated that the first 40 amino acids were identical to the ribosomal L5 protein, found in the large rRNA subunit and bound to 5S RNA. Partial peptide maps of the authentic L5 protein and the 34,000-molecular-weight protein were identical. mdm-2-L5 and mdm-2-L5-p53 complexes were shown to bind 5S RNA specifically, presumably through the known specificity of L5 protein for 5S RNA. In 5S RNA-L5-mdm-2-p53 ribonucleoprotein complexes, it was also possible to detect the 5.8S RNA which has been suggested to be covalently linked to a percentage of the p53 protein in a cell. These experiments have identified a unique ribonucleoprotein complex composed of 5S RNA, L5 protein, mdm-2 proteins, p53 protein, and possibly the 5.8S RNA. While the function of such a ribonucleoprotein complex is not yet clear, the identity of its component parts suggests a role for these proteins and RNA species in ribosomal biogenesis, ribosomal transport from the nucleus to the cytoplasm, or translational regulation in the cell.     

Small protein B interacts with the large and the small subunits of a stalled ribosome during trans-translation

During trans-translation, stalled bacterial ribosomes are rescued by small protein B (SmpB) and by transfer-messenger RNA (tmRNA). Stalled ribosomes switch translation from the defective messages to a short internal reading frame on tmRNA that tags the nascent peptide chain for degradation and recycles the ribosomes. We present evidences that SmpB binds the large and small ribosomal subunits in vivo and in vitro. The binding between SmpB and the ribosomal subunits is very tight, with a dissociation constant of 1.7 × 10⁻¹⁰ M, similar to its K_D for the 70S ribosome or for tmRNA. tmRNA displaces SmpB from its 50S binding but not from the 30S. In vivo, SmpB is detected on the 50S when trans-translation is impaired by lacking tmRNA or a functional SmpB. SmpB contacts the large subunit transiently and early during the trans-translational process. The affinity of SmpB for the two ribosomal subunits is modulated by tmRNA in the course of trans-translation. It is the first example of two copies of the same protein interacting with two different functional sites of the ribosomes.     

Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.     

MRPS27 is a pentatricopeptide repeat domain protein required for the translation of mitochondrially encoded proteins

Mammalian pentatricopeptide repeat domain (PPR) proteins are involved in regulation of mitochondrial RNA metabolism and translation and are required for mitochondrial function. We investigated an uncharacterised PPR protein, the supernumerary mitochondrial ribosomal protein of the small subunit 27 (MRPS27), and show that it associates with the 12S rRNA and tRNA^Glu, however it does not affect their abundance. We found that MRPS27 is not required for mitochondrial RNA processing or the stability of the small ribosomal subunit. However, MRPS27 is required for mitochondrial protein synthesis and its knockdown causes decreased abundance in respiratory complexes and cytochrome c oxidase activity.

Structured summary of protein interactions

MRPS27 and MRPS15 colocalize by cosedimentation through density gradient (View Interaction)     

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

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.