RNA sequences in ribonucleoprotein fragments of the complex formed from ribosomal 23-S RNA and ribosomal protein L24 of Escherichia coli.

Upon digestion of the complex formed from the 23-S ribosomal RNA and the 50-S ribosomal protein L24 of Escherichia coli, two fragments resistant to ribonuclease were recovered; these fragments contained RNA sections belonging to the 480 nucleotides at the 5' end of 23-S RNA. By determining the sequence of 70% of this latter region we were able to localise the sections which, in the presence of the protein, are resistant to ribonuclease. Our results suggest that the region encompassing the 480 nucleotides starting at the 9th nucleotide from the 5' end of 23-S RNA has a compact tertiary structure, which is stabilised by protein L24.     

Binding of thiostrepton to a complex of 23-S rRNA with ribosomal protein L11.

Thiostrepton binds with high affinity and with a 1 : 1 stoichiometry to a complex formed between Escherichia coli 23-S ribosomal RNA and ribosomal protein L11 of E. coli or the homologous protein BM-L11 of Bacillus megaterium. In the presence of T1 ribonuclease, protein BM-L11 and thiostrepton protect from degradation a fragment of E. coli 23-S RNA estimated to be about 50 nucleotides in length.     

On the distribution and packing of RNA and protein ribosomes.

From the analysis of the measured radii of gyration of the RNA (Rg = 6.6 +/- 0.3 nm) and protein (Rg = 10.2 +/- 0.5 nm) components of the 50-S subparticle of Escherichia coli ribosomes it is concluded that proteins containing a large amount of hydrodynamically bound water are located on the periphery of the tightly packed RNA. We found that the common features of the measured X-ray scattering curves of the E. coli 70-S ribosome, its 30-S and 50-S subparticles and wheat 80-S ribosomes in the region of scattering angles corresponding to scattering vectors mu from 1 to 5 nm-1 reflect features of the RNA compact packing. A hypothesis is proposed that the compact packing of RNA helices in the range of Bragg distances of 4.5--2.0 nm is a general structural feature of all ribosomal particles.     

Small-angle X-ray titration study on the complex formation between 5-S RNA and the L18 protein of the Escherichia coli 50-S ribosome particle.

The 5-S RNA (A) and the L18 protein (B) from Escherichia coli ribosomes form one single AB complex in the concentration ranges supposed to prevail in vivo; at concentrations of L18 higher than 40 mM there is some indication for a minor species, most probably an AB2 species. This is indicated from the X-ray scattering titration data of the 5-S RNA/L18 system recorded at 21 degrees C in ribosomal reconstitution buffer. As a result of the 1:1 complex formation, there is a relatively small but defined increase in the radius of gyration from 3.61 to 3.85 nm. This result as well as the experimental scattering curve can be explained by models where it is assumed that the elongated L18 model is quite far from the electron density centre and where protein L18 interacts with one or both of the minor arms of the supposed Y-shaped 5-S RNA molecule.     

The ternary 5-S RNA complex of proteins L 18 and L 25. A small-angle X-ray scattering titration study.

The 5-S RNA (A) and the proteins L 18 (B) and L25 (C) from Escherichia coli ribosomes form a ternary complex of the type ABC with a stepwise stability constant, log K111 approximately equal to 6.5. This is indicated from X-ray scattering titrations recorded at 21 degrees C in ribosomal reconstitutional buffer. When the ternary ABC complex forms there is only a limited change in the scattering curve compared to that of 5-S RNA, indicating that 5-S RNA does not undergo a major conformational change during the complex formation. The increase in the radius of gyration from 3.61 nm (5-S RNA) to 3.95 nm (ABC complex) as well as the experimental scattering curve can be explained by models where it is assumed that the elongated L 18 and L25 models are quite far from the electron density centre and where the protein molecules interact mainly with the minor arms of the supposed Y-shaped 5-S RNA molecule.     

The topography of the 5' end of 16-S RNA in the presence and absence of ribosomal proteins S4 and S20

A ribonucleoprotein prepared by strong ribonuclease digestion of a complex of 16-S ribosomal RNA and proteins S4 and S20 from Escherichia coli has been characterized; its nucleotide sequence, the positions of enzyme cuts and the sequence excisions have been placed in the completed sequence of 16-S RNA. The positions and yields of enzyme cuts, and excisions of sequence, are compared with those of various ribonucleoproteins prepared with S4 or S20 alone, and with the ribonuclease-resistant S4 RNA prepared from renatured 16-s RNA in the absence of ribosomal protein. These data yield important information on the topography and organisation of the 5' third of the 16-s RNA which is selectively maintained in its native conformation by the bound proteins; they also provide criteria for testing secondary structural models of this region of 16-S RNA.     

The composition and properties of the Escherichia coli 5-S RNA-protein complex

At a high concentration of MgCl2 (30 mM) and a low concentration of proteins from the 50-S subunit (0.2 mg/ml), only three proteins, L15, L18 and L25, bind to 5-S RNA in significant amounts. On the other hand, in a buffer containing only 1 mM Mg Cl2, but otherwise at the same ionic strength (0.2 M), or at a protein concentration about 1.5 mg/ml, a large, stable complex can form between immobilized 5-S RNA and 50-S ribosomal proteins. This complex contains proteins L2, L3, L5, L15, L16, L17, L18, L21, L22, L25, L33 and L34, and it possess properties relevant to the function of the 50-S subunit; it has a binding site for deacylated tRNA, with a dissociation constant of 4.5 x 10(-7) M. The complex formed with 5-S RNA immobilized on an affinity column interacts also with 30-S subunits. The 5-S RNA-protein complex is interpreted as a sub-ribosomal domain which includes a considerable fraction of the peptidyl transferase center of the Escherichia coli ribosome.     

Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

The ribosomes from four temperature-sensitive mutants of Escherichia coli have been examined for defects in cell-free protein synthesis. The mutants examined had alterations in ribosomal proteins S10, S15, or L22 (two strains). Ribosomes from each mutant showed a reduced activity in the translation of phage MS2 RNA at 44 degrees C and were more rapidly inactivated by heating at this temperature compared to control ribosomes. Ribosomal subunits from three of the mutants demonstrated a partial or complete inability to reassociate at 44 degrees C. 70-S ribosomes from two strains showed a reducton in messenger RNA binding. tRNA binding to the 30 S subunit was reduced in the strains with altered 30-S proteins and binding to the 50 S subunit was affected in the mutants with a change in 50 S protein L22. The relation between ribosomal protein structure and function in protein synthesis in these mutants is discussed.     

Characterization of the Escherichia coli 23S Ribosomal RNA region associated with ribosomal protein L1. Evidence for homologies with the 5''-end region of the L11 operon.

The results previously obtained upon studying the L1-23S RNA complex by the fingerprint technique have been reexamined in the light of new data on 23S RNA primary structure. The 23S RNA region that remains associated with the L1 ribosomal protein after RNase digestion of the synthetic complex lies between nucleotides 2067 and 2235 from the 5'-end of the molecule. This region contains a m7G near to the 5'-end and possesses a high degree of mutability in E. coli. Three different sequences were observed in E. coli MRE 600. All three sequences differ in two positions relative to the corresponding sequence in rrnB cistron from E. coli K12. Striking homology is observed between the 23S RNA region associated with protein L1 and the 5'-part of L11 operon. This observation supports the model of feedback regulation of r-proteins synthesis proposed by Yates et al. (PNAS, 77, 1837) and strongly suggests that the region of 23S RNA located between positions 2155 and 2202 is essential for the binding of protein L1.     

The organization of ribosomal RNA genes in the mitochondrial DNA of Tetrahymena pyriformis strain ST.

1. We have constructed a physical map of the mtDNA of Tetrahymena pyriformis strain ST using the restriction endonucleases EcoRI, PstI, SacI, HindIII and HhaI. 2. Hybridization of mitochondrial 21 S and 14 S ribosomal RNA to restriction fragments of strain ST mtDNA shows that this DNA contains two 21-S and only one 14-S ribosomal RNA genes. By S1 nuclease treatment of briefly renatured single-stranded DNA the terminal duplication-inversion previously detected in this DNA (Arnberg et al. (1975) Biochim. Biophys. Acta 383, 359--369) has been isolated and shown to contain both 21-S ribosomal RNA genes. 14 S ribosomal RNA hybridizes to a region in the central part of the DNA, about 8000 nucleotides or 20% of the total DNA length apart from the nearest 21 S ribosomal RNA gene. 3. We have confirmed this position of the three ribosomal RNA genes by electron microscopical analysis of DNA . RNA hybrid molecules and R-loop molecules. 4. Hybridization of 21 S ribosomal RNA with duplex mtDNA digested either with phage lambda-induced exonuclease or exonuclease III of Escherichia coli, shows that the 21-S ribosomal RNA genes are located on the 5'-ends of each DNA strand. Electron microscopy of denaturated mtDNA hybridized with a mixture of 14-S and 21-S ribosomal RNAs show that the 14 S ribosomal RNA gene has the same polarity as the nearest 21 S ribosomal RNA gene. 5. Tetrahymena mtDNA is (after Saccharomyces mtDNA) the second mtDNA in which the two ribosomal RNA cistrons are far apart and the first mtDNA in which one of the ribosomal RNA cistrons is duplicated.     

Analysis of proteinase A function in yeast

The antibiotic, micrococcin, binds to complexes formed between bacterial 23-S ribosomal RNA and ribosomal protein L11 and, in doing so, inhibits of thiostrepton. In assay systems simulating partial reaction of protein synthesis, micrococcin inhibits a number of processes believed to involve the ribosomal A site while stimulating GTP hydrolysis dependent upon ribosomes and elongation factor EF-G. The latter effect is not observed upon ribosomes lacking a protein homologous with protein L11. Nor is it apparent upon those containing 23-S RNA previously subjected to the action of a specific methylase known to render ribosomes resistant to thiostrepton. It is concluded that stimulation by micrococcin of factor-dependent GTP hydrolysis results from the binding of the drug to its normal target site which involves 23-S RNA and protein L11.     

The 5-S RNA . protein complex from an extreme halophile, Halobacterium cutirubrum. Purification and characterization.

A 5-S RNA . protein complex has been isolated from the 50-S ribosomal subunit of an extreme halophile, Halobacterium cutirubrum. The 50-S ribosomal subunit from the extreme halophile requires 3.4 M K+ and 100 mM Mg2+ for stability. However, if the high K+ concentration is maintained but the Mg2+ concentration lowered to 0.3 mM, the 5-S RNA . protein complex is selectively extracted from the subunit. After being purified on an Agarose 0.5-m column the complex had a molecular weight of about 80000 and contained 5-S RNA and two proteins, HL13 and HL19, with molecular weights (by sedimentation equilibrium) of 18700 and 18000, respectively. No ATPase or GTPase activity could be detected in the 5-S RNA . protein complex. The amino acid composition and electrophoretic mobility on polyacrylamide gels indicated both proteins were much more acidic than the equivalent from Escherichia coli or Bacillus stearothermophilus. Partial amino acid sequence data suggest HL13 is homologous to EL18 and HL19 to EL5.     

The synthesis of a photoreactive puromycin analogue and its application for labeling proteins in the 50-S subunit of Escherichia coli ribosomes.

A photoreactive puromycin analogue, 6-dimethylamino-9-[3-(p-azido-L-beta-phenylalanylamino)-3-deoxy-beta-ribofuranosyl] purine, was synthesized. Biological activity was demonstrated by inhibition of the poly (U)-directed phenylalanine-incorporation system and by decomposition of isolated polysomes from Escherichia coli. The 3H-labeled puromycin analogue was covalently attached to the 50-S subunit of isolated 70-S ribosomes from Escherichia coli after irradiation. More than 90% of the radioactivity was bound to the protein fraction. The 70-S proteins were separated by two-dimensional gel electrophoresis. The proteins labeled primarily were those of the 50-S subunit, identified as L6, L13, L18, L22 and L25. On the basis of the affinity label used and supportive data from the literature, it is concluded that these proteins are at the active center of the 50-S particle and probably belong to the region of the ribosomal A site.     

Defect in the split proteins of 30-S ribosomal subunits and under-methylation of 16-S ribosomal RNA in a polyamine-requiring mutant of Escherichia coli grown in the absence of polyamines.

Polyphenylalanine synthesis was carried out with Escherichia coli Q13 50-S ribosomal subunits and reconstituted 30-S particles containing different combinations of 23-S core particles and 30-S subunit split proteins obtained from a polyamine-requiring mutant of E. coli during its growth in the presence or absence of putrescine. It was concluded that the defect in the amount of some kinds of 30-S subunit split proteins was responsible for the decrease of polypeptide synthesis in a polyamine-requiring mutant of E. coli grown in the absence of polyamines. The methylation of 16-S RNA during growth in the absence of putrescine was decreased, while the degree of methylation of 23-S RNA did not change significantly. The decrease in methylation of 16-S RNA in the absence of putrescine was due mainly to a decrease of methylation of adenine. The relationship between the decrease of polypeptide synthetic activity of 30-S ribosomal subunits obtained from a polyamine-requiring mutant of E. coli grown in the absence of polyamines and the decrease of methylation of 16-S RNA is discussed.     

Cold-sensitive ribosome assembly in an Escherichia coli mutant lacking a single methyl group in ribosomal protein L3

Ribosomal protein methylation has been well documented but its function remains unclear. We have examined this phenomenon using an Escherichia coli mutant (prmB2), which fails to methylate glutamine residue number 150 of ribosomal protein L3. This mutant exhibits a cold-sensitive phenotype: its growth rate at 22 degrees C is abnormally low in complete medium. In addition, strains with this mutation accumulate abnormal and unstable ribosomal particles; 50-S and 30-S subunits are formed, but at a lower rate. Once assembled, ribosomes with unmethylated L3 are fully active by several criteria. (a) Protein synthesis in vitro with purified 70-S prmB2 ribosomes is as active as wild-type using either a natural (R17) or an artificial [poly(U)] messenger. (b) The induction of beta-galactosidase in vivo exhibits normal kinetics and the enzyme has a normal rate of thermal denaturation. (c) These ribosomes are standard when exposed in vitro to a low magnesium concentration or increasing molarities of LiCl. Efficient methylation of L3 in vitro requires either unfolded ribosomes or a mixture of ribosomal protein and RNA. We suggest that the L3-specific methyltransferase may qualify as one of the postulated 'assembly factors' of the E. coli ribosome.     

Ribosomal proteins at the stalk region modulate functional rRNA structures in the GTPase center

Replacement of the L10.L7/L12 protein complex and L11 in Escherichia coli ribosomes with the respective rat counterparts P0.P1/P2 and eukaryotic L12 causes conversion of ribosomal specificity for elongation factors from prokaryotic elongation factor (EF)-Tu/EF-G to eukaryotic EF (eEF)-1alpha/eEF-2. Here we have investigated the effects of protein replacement on the structure and function of two rRNA domains around positions 1070 and 2660 (sarcin/ricin loop) of 23 S rRNA. Protein replacement at the 1070 region in E. coli 50 S subunits was demonstrated by chemical probing analysis. Binding of rat proteins to the 1070 region caused increased accessibility of the 2660 and 1070 regions to ligands for eukaryotic ribosomes: the ribotoxin pepocin for the 2660 region (E. coli numbering), anti-28 S autoantibody for the 1070 region, and eEF-2 for both regions. Moreover, binding of the E. coli L10.L7/L12 complex and L11 to the 1070 region was shown to be responsible for E. coli ribosomal accessibility to another ribotoxin, gypsophilin. Ribosomal proteins at the 1070 region appear to modulate the structures and functions of the 2660 and 1070 RNA regions in slightly different modes in prokaryotes and eukaryotes.     

Nucleotide sequence of cloned cDNA specific for rat ribosomal protein L35a

A cDNA clone specific for rat ribosomal protein L35a, which is known to be a tRNA-binding protein, was isolated by hybrid-selected translation from a cDNA library made for 8-9-S poly(A)-rich RNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. It consists of one base pair from the 5' leading sequence, the entire coding sequence of 333 base pairs and 14 base pairs from the 3' trailing sequence. The primary structure of protein L35a was deduced from the nucleotide sequence. It consists of 109 amino acids with a molecular mass of 12422. The calculated amino acid composition is consistent with that reported for the hydrolysate of L35a. The amino acid sequence showed marked homology with the reported partial sequence of Xenopus leavis ribosomal protein L32, but not significant homology with Escherichia coli ribosomal proteins that bind to tRNA.     

The effect of a cytidine-to-uridine transition on the stability of Escherichia coli A19 5-S RNA

We have been able to isolate several species of 5-S ribosomal RNA from Escherichia coli A19. These molecules were separated on the basis of their differing stabilities during electrophoresis on 12% polyacrylamide gels in 7 M urea. This differing stability is shown, in one case, to be due to a different primary sequence. We have determined the sequence of the least stable of these molecules and have found only one difference to the published sequence of E. coli A19 5-S RNA, namely a uridine in place of a cytidine at position 92. The consequent G x U base pair, formed in a normally highly stable G x C-rich region, is responsible for a drastic reduction in the stability of the molecule. This instability leads to a less constrained, more compact molecule which thus migrates faster in electrophoresis under denaturing conditions. This species of 5-S RNA is shown to make up 30% of the total 5-S RNA in the 50-S ribosomal subunits in this organism. Further structural studies were carried out using S1 nuclease digestion, sodium bisulphite modification and thermal melting analysis. All these methods indicate a 5-S RNA drastically destabilized in parts of its secondary and tertiary structure. Finally, the ability of the variant 5-S RNA to recognize and form a complex with its 50-S subunit binding proteins was examined and found to be impaired.