RIBOSOMAL RNA ON THE SURFACE OF RIBOSOMES

Ribonuclease from pancreas releases nucleotide from Escherichiacoli ribosomes while not altering significantly the base composition of the total ribosomal RNA. Ribonuclease hydrolyzes ribsomal RNA without destroying the structure of 70S or 30S and 50S ribosomes. The RNA in 30S and 50S ribosomes appears more sensitive to the action of ribonuclease. The data suggest that RNA may be a surface component of the ribosome.     

Cytoplasmic and chloroplast ribosomes of Chlamydomonas: ultracentrifugal characterization

Ribosomes isolated from the cytoplasmic and chloroplast fractions of Chlamydomonas were characterized in the ultracentrifuge. The cytoplasmic ribosomes belong to the 80S class of ribosomes, and, like animal ribosomes, dissociate to 60, 50, and 40S subunits. However, like the ribosomes of microorganisms, they contain smaller RNA's, 24 and 16S, and require 0.01 mole of magnesium ions per liter for stability. Chloroplast ribosomes are 70S like those of higher plants but are very unstable. A stable 50S subunit has been observed.     

Heterogeneity of 5S RNA in fungal ribosomes

Neurospora crassa has at least seven types of 5S RNA genes (alpha, beta, gamma, epsilon, delta, zeta, and eta) with different coding regions. A high resolution gel electrophoresis system was developed to separate minor 5S RNA's from the major 5S RNA (alpha). A study of several Neurospora crassa strains, four other species in the genus Neurospora, members of two closely related genera, and three distantly related genera demonstrated that 5S RNA heterogeneity is common among fungi. In addition, different 5S RNA's are present in Neurospora ribosomes. The finding that fungal ribosomes are structurally heterogeneous suggests that ribosomes may be functionally heterogeneous as well.     

How hibernation factors RMF, HPF, and YfiA turn off protein synthesis

Eubacteria inactivate their ribosomes as 100S dimers or 70S monomers upon entry into stationary phase. In Escherichia coli, 100S dimer formation is mediated by ribosome modulation factor (RMF) and hibernation promoting factor (HPF), or alternatively, the YfiA protein inactivates ribosomes as 70S monomers. Here, we present high-resolution crystal structures of the Thermus thermophilus 70S ribosome in complex with each of these stationary-phase factors. The binding site of RMF overlaps with that of the messenger RNA (mRNA) Shine-Dalgarno sequence, which prevents the interaction between the mRNA and the 16S ribosomal RNA. The nearly identical binding sites of HPF and YfiA overlap with those of the mRNA, transfer RNA, and initiation factors, which prevents translation initiation. The binding of RMF and HPF, but not YfiA, to the ribosome induces a conformational change of the 30S head domain that promotes 100S dimer formation.     

DNA Complementary to Ribosomal RNA: Relation between Genomic Proportion and Ploidy

Ten Nicotiana species were assayed for the proportion of DNA that is complementary to ribosomal RNA. This proportion varies from 0.27 to 0.9 percent, with tetraploid species having lower values than the diploid species. The tetraploid species have about twice as much DNA per cell as do diploid species. Thus, the absolute number of genes for ribosomal RNA varies less than the proportion of complementary DNA. Further, the number of genes for the RNA in 80S ribosomes varies less among species than does that for the RNA in 70S ribosomes. The data indicate that loss of DNA complementary to ribosomal RNA is associated with tetraploidy in the genus Nicotiana.     

Ribosomal Protein Conferring Sensitivity to the Antibiotic Spectinomycin in Escherichia coli

Reconstitution of 30S ribosomal particles from 16S ribosomal RNA and total proteins, or from core proteins and split proteins obtained from the ribosomes of strains of Escherichia coli sensitive to and resistant to spectinomycin, shows that the split protein fraction determines the response of polypeptide synthesis in virto to spectinomycin. Reconstitution of active particles in the presence of isolated split proteins allowed the identification of the single split protein responsible for spectinomycin sensitivity.     

Crystal structure of the ribosome at 5.5 A resolution

We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.     

Ribosomes: a common structural feature

A reflection between 45 and 50 A has been observed in x-ray diffraction patterns from ribosomes extracted from Escherichia coli, Drosophila larvae, rat liver, and rabbit reticulocytes. This spacing appears to correspond to a common substructural feature within the ribosomes. The intensity distribution is consistent with a model in which part of the RNA is in the form of four or five parallel double helices 45 to 50 A apart.     

Structural basis for the rescue of stalled ribosomes: structure of YaeJ bound to the ribosome

In bacteria, the hybrid transfer-messenger RNA (tmRNA) rescues ribosomes stalled on defective messenger RNAs (mRNAs). However, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmRNA-independent manner. Here, we report a 3.2 angstrom-resolution crystal structure of the rescue factor YaeJ bound to the Thermus thermophilus 70S ribosome in complex with the initiator tRNA(i)(fMet) and a short mRNA. The structure reveals that the C-terminal tail of YaeJ functions as a sensor to discriminate between stalled and actively translating ribosomes by binding in the mRNA entry channel downstream of the A site between the head and shoulder of the 30S subunit. This allows the N-terminal globular domain to sample different conformations, so that its conserved GGQ motif is optimally positioned to catalyze the hydrolysis of peptidyl-tRNA. This structure gives insights into the mechanism of YaeJ function and provides a basis for understanding how it rescues stalled ribosomes.     

Structure of Ribosomal Subunits of M. vannielii: Ribosomal Morphology as a Phylogenetic Marker

On the basis of ribosomal morphology, it has been proposed that the sulfur-metabolizing archaebacteria constitute a group (the eocytes) with a phylogenetic importance equal to that of the eubacteria, archaebacteria, and eukaryotes. It has been further proposed that eocytes should be given kingdom status. Ribosomal subunits from the methanogenic archaebacterium Methanococcus vannielii were examined by electron microscopy, and their structures were compared to those of other archaebacterial, eubacterial, and eukaryotic ribosomes. 30S subunits from M. vannielii showed the elongated contour and the one-third to two-thirds partition characteristic of such subunits. In addition, the angled asymmetric projections of those subunits showed a squarish base and a beak on the head. 50S subunits from M. vannielii were seen in both crown and kidney views. In crown views, the L1 protuberance was frequently pronounced and split; an incision below this protuberance and a protrusion at the base of the particle were also observed. Although previous studies suggested that certain of these structural features were found exclusively in ribosomes from sulfur-metabolizing archaebacteria, these new results indicate that such features also occur in ribosomes from a typical methanogenic archaebacterium and thus may not be reliable phylogenetic markers.     

Secondary structure of ribosomal RNA

Infrared spectra were obtained for 16S and for 23S ribosomal RNA's in D(2)O solutions. The percentage of each base in the paired and unpaired regions of the RNA was determined from the spectra. The secondary structures of 16S and 23S ribosomal RNA's (from Escherichia coli) are significantly different from each other and are also different from those of yeast ribosomal RNA, formylmethionyl-transfer RNA, and the anticodon fragment of this transfer RNA.     

Functional chloroplast polyribosomes from tobacco leaves

Incubation of isolated to bacco chloroplasts with ingredients re quired for protein synthesis resulted in liberation of 70S ribosomnes and poly ribosomes that no longer sedimented with the chloroplasts. With increasing time of incubation, polyribosomes broke down to 70S monosomes. Similarly, microgram quantities of ribonuclease caused chloroplast polyribosomes to break down into monosomes. Both polyribosomes and 70S ribosomes that were isolated on sucrose density gra dients and tested separately in cell-free systems were capable of protein syn thesis; however, polyribosomes formed more protein per unit of RNA than monosomes did.     

5S RNA synthesized by Escherichia coli in presence of chloramphenicol: different 5'-terminal sequences

Escherichia coli cells, grown in the presence of chloramphenicol, synthesize a low molecular weight RNA (CM-5S RNA) not bound to ribosomes which is similar to ribosomal 5S RNA. Oligonucleotide patterns derived from ribonuclease digests of 5S RNA and of CM-5S RNA are indistinguishable except that the 5'-terminal oligonucleotides differ. Whereas the nucleotide sequence of the 5'-terminus of normal 5S RNA is (p)U(p)G-, there are three alternate sequences of the 5'-terminus of CM-5S RNA: (p)U(p)U(p)G-, (p)U(p)U(p)U(p)G-, and (p)A(p)U(p)U(p)U(p)G-.     

Ribosomes: effect of interferon on their interaction with rapidly labeled cellular and viral RNA's

Rapidly labeled RNA of mouse L cells and labeled RNA of Mengo virus, unlike cellular RNA labeled under steady-state conditions, form detectable complexes with L-cell ribosomes. These ribosome-RNA complexes formed in vitro appear analogous to those assembled during polysome formation in vivo. When ribosomes are prepared from L cells exposed to homologous interferon, their capacity to associate with cell messenger is preserved, while their ability to interact with viral RNA is markedly reduced. The ribosomes from cells exposed to interferon are thus altered selectively to permit only certain messages to be bound and translated.     

Inhibition of protein synthesis by spectinomycin

Spectinomycin selectively inhibits protein synthesis in cells and in extracts of Escherichia coli. Mutations to high-level resistance to this antibiotic map close to the streptomycin locus, and the site of action of spectinomycin, like that of streptomycin, is the 30S ribosomal subunit, as shown by experiments with reconstituted 70S ribosomes containing subunits from sensitive and from resistant ribosomes. In contrast to streptomycin, however, spectinomycin is not bactericidal and causes no detectable misreading of polyribonucleotides.     

Erythromycin-resistant mutant of Escherichia coli with altered ribosomal protein component

Erythromycin combines with 50S ribosomal subunit of an erythromycin-sensitive Escherichia coli (strain Q13), while ribosomes from an erythromycin-resistant mutant from this strain have little affinity for the antibiotic. A protein component of the 50S subunit of the mutant strain is distinct from that of the parent Q13 strain.     

Altered ribosomal protein in streptomycin-dependent Escherichia coli

We have compared the 30S ribosomal proteins of strains of Escherichia coli sensitive to and dependent on streptomycin and identified a single protein that is functionally altered in the ribosomes dependent on streptomycin. This protein (30S-15) is the same protein that is functionally altered in ribosomes resistant to streptomycin.     

Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6

Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.     

Reversal of creatine kinase translational repression by 3' untranslated sequences

A subline of U937 cells (U937D) was obtained in which creatine kinase B (CK-B) messenger RNA was present and bound to ribosomes, but CK activity was undetectable. Transformation of U937D cells with retrovirus vectors that contain the 3' untranslated region (3' UTR) of CK-B messenger RNA exhibited CK activity with no change in abundance of CK-B mRNA. The 3' UTR formed a complex in vitro with a component of S100 extracts from wild-type cells. This binding activity was not detectable in S100 extracts from cells that expressed CK activity after transformation with the 3' UTR-containing vector. These results suggest that translation of CK-B is repressed by binding of a soluble factor or factors to the 3' UTR.     

Detergent-solubilized RNA polymerase from cells infected with foot-and-mouth disease virus

The foot-and-mouth disease virus RNA polymerase complex was dissociated from cellular membranes with deoxycholate in the presence of dextran sulfate. The soluble polymerase complex was active in the cell-free synthesis of virus-specific RNA; solubilization of the complex permitted direct analysis of the cell-free reaction mixtures without recourse to RNA extraction. A major RNA-containing component found early during cell-free incubation ranged from approximately 140 to 300S. The final major products of the cell-free system were 37S virus RNA, 20S ribonuclease-resistant RNA, and a 50S component containing RNA.