Primary structure of protein L19 from the large subunit of Escherichia coli ribosomes.

Protein L19, a component of the Escherichia coli 50S ribosomal subunit implicated in 30S-50S subunit interaction was sequenced by the dansyl-Edman method. L19 consists of a single polypeptide chain of 114 amino acids giving a calculated molecular weight of 13 002. Peptides obtained from various enzymatic cleavages were isolated on thin-layer peptide maps or gel filtration. Automated Edman degradation using a liquid phase sequenator was carried out on the whole protein as well as on a large 58-residue fragment arising from digestion with Staphylococcus aureus protease. Every position in protein L19 was confirmed at least twice. Results of secondary structure estimation and homologies with other E. coli ribosomal protein sequences are presented.     

Mapping proteins of the 50S subunit from Escherichia coli ribosomes

Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui.     

Three-dimensional structure of the large ribosomal subunit from Escherichia coli.

The three-dimensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens. A new method of three-dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle. A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit. This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.     

50-S subunit from Escherichia coli ribosomes. Isolation of active ribosomal proteins and protein complexes.

A method is described for the isolation of highly purified proteins from the 50-S subunit of Escherichia coli ribosomes. All the proteins from the large subunit could be isolated with the exception of L14, L26, L31 and L34. The isolated proteins are functionally active in reconstituted particles. The method consists of successive NH4Cl/EtOH and LiCl washing steps, which split off distinct groups of proteins from the ribosome. The protein groups are further separated by a combination of gel filtration (Sephadex G-100) and ion-exchange chromatography (carboxymethylcellulose) in the presence of 6 M urea, at neutral pH and 4 degrees C. The purity of the proteins was analyzed by two-dimensional gel electrophoresis. In addition, ten protein complexes were isolated and identified.     

Secondary structure of the large subunit ribosomal RNA from Escherichia coli, Zea mays chloroplast, and human and mouse mitochondrial ribosomes.

Short base-paired RNA fragments, and fragments containing intra-RNA cross-links, were isolated from E. coli 23S rRNA or 50S ribosomal subunits by two-dimensional gel electrophoresis. The interactions thus found were used as a first basis for constructing a secondary structure model of the 23S rRNA. Sequence comparison with the 23S rDNA from Z. mays chloroplasts, as well as with the 16S (large subunit) rDNA from human and mouse mitochondria, enabled the experimental model to be improved and extrapolated to give complete secondary structures of all four species. The structures are organized in well-defined domains, with over 450 compensating base changes between the two 23S species. Some ribosomal structural "'switches" were found, one involving 5S rRNA.     

Unfolding of E. coli ribosomes: evidence for pre-existing breaks in the large subunit.

An investigation has been made on structure modifications of E. coli ribosomes following EDTA treatment. When completely deprived of magnesium, the small subunit sediments at 16S while the large one, in the same conditions, shows two components at 17S and 21S. Unfolding causes in both subunits an increase in radius of gyration without substantial change in molecular weight, as shown by light scattering measurements. The occurence of the slower 17S component besides the 21S one has to be connected with a fraction of the large subunit population which presents nucleolytic breaks in its RNA chain. These breaks do not cause fragmentation of the unfolded subunit but lead to a more open configuration sedimenting at lower velocity.     

RNA chaperone activity of large ribosomal subunit proteins from Escherichia coli

The ribosome is a highly dynamic ribonucleoprotein machine. During assembly and during translation the ribosomal RNAs must routinely be prevented from falling into kinetic folding traps. Stable occupation of these trapped states may be prevented by proteins with RNA chaperone activity. Here, ribosomal proteins from the large (50S) ribosome subunit of Escherichia coli were tested for RNA chaperone activity in an in vitro trans splicing assay. Nearly a third of the 34 large ribosomal subunit proteins displayed RNA chaperone activity. We discuss a possible role of this function during ribosome assembly and during translation.     

Localization of sites of photoaffinity labeling of the large subunit of Escherichia coli ribosomes by arylazide derivative of puromycin

Previous work (Nicholson, A. W., Hall, C. C., Strycharz, W. A., and Cooperman, B. S. (1982) Biochemistry 21, 3797-3808) showed that [3H]p-azidopuromycin photoaffinity labeled 70 S Escherichia coli ribosomes and that photoincorporation into 50 S subunit proteins was in the order L23 greater than L18/22 greater than L15. In the present work we report on immunoelectron microscopic studies of the complexes formed by p-azidopuromycin-modified 50 S subunits with antibodies to the N6,N6-dimethyladenosine moiety of the antibiotic. The p-azidopuromycin-modified 50 S subunits appear to be identical to unmodified control subunits in electron micrographs. Complexes of modified subunits with antibodies to the N6,N6-dimethyladenosine moiety of p-azidopuromycin were visualized in micrographs. Individual subunits with a single bound antibody (monomeric complexes) and pairs of subunits cross-linked by a single antibody (dimeric complexes) were separately evaluated and showed similar results. Two regions of p-azidopuromycin photoincorporation were identified. The primary site, seen in about 75% of the complexes, is between the central protuberance and small projection, on the side away from the L7/L12 arm, in a region thought to contain the peptidyltransferase center. The secondary site, of unknown significance, is at the base of the subunit maximally distant from the arm. These placements are essentially identical to those we observed in analyses of puromycin photoincorporation (Olson, H. M., Grant, P. G., Cooperman, B. S., and Glitz, D. G. (1982) J. Biol. Chem. 257, 2649-2656) and quantitatively similar to evaluations of monomeric puromycin-50 S subunit complexes. The data support the placement of proteins L23, L18/22, and L15 at or near the peptidyltransferase center at the primary site and suggest, in addition, that the secondary site includes a genuine area of puromycin affinity.     

Programmed ribosomal frameshifting generates the Escherichia coli DNA polymerase III gamma subunit from within the tau subunit reading frame.

The Escherichia coli dnaX gene encodes both the tau and gamma subunits of DNA polymerase III holoenzyme in one reading frame. The 71.1 kDa tau and the shorter gamma share N-terminal sequences. Mutagenesis of a potential ribosomal frameshift signal located at codons 428-430 without changing the amino acid sequence of the tau product, eliminated detectable synthesis of the gamma subunit, suggesting that the reading frame is shifted at that sequence and gamma is terminated by a nonsense codon located in the -1 frame 3 nucleotides downstream of the signal. This seems to be the first known case of a frameshift which is used, along with the termination codon in the -1 frame, to terminate a peptide within a reading frame. [Mutagenesis of a dibasic peptide (lys-lys) at codons 498-499, the site at which a tau'-'LacZ fusion protein was cleaved in vitro (1) had no effect on gamma formation in vivo, suggesting that cleavage observed in vitro is not the mechanism of gamma formation in vivo.     

In vivo studies on the incorporation of microinjected acidic proteins of the large ribosomal subunit from Escherichia coli and artermia salina into oocyte ribosomes from Xenopus laevis.

Tritium-labeled acidic proteins from the large ribosomal subunit of Artermia salina or Escherichia coli were microinjected into the cytoplasm of stage IV/V oocytes from Xenopus laevis. eL12 from the large ribosomal subunit of A. salina but not L7/L12 or L7/L12--L10 from E. coli is specifically incorporated into 60S ribosomal subunits of oocytes. This incorporation is not significantly inhibited by actinomycin D. Incorporation of eL12 into the 60S subunits occurs in enucleated oocytes, suggesting that active ribosomal ribonucleic acid synthesis and ribosome assembly as well are not prerequired for this reaction. Apparently the incorporation proceeds via an exchange reaction between a free cytoplasmic pool of eL12 and ribosomal eL12.     

Probing the function of conserved RNA structures in the 30S subunit of Escherichia coli ribosomes.

Ribosomes play an active role in protein biosynthesis. Ribosomal RNA conformation in ribosomal subunits, intramolecular interactions between different rRNA sequences within the confinement of the particles, and intermolecular interactions are presumed necessary to support efficient and accurate protein synthesis. Here we report an analysis of the disposition of 16S rRNA conserved zones centered about positions 525, 1400, and 1500 in 30S subunits. Complementary oligodeoxyribonucleotides in conjunction with nuclease S1 digestion were used to do this. All of the sequences examined in 30S subunits are accessible to DNA probes of 9 to 12 nucleotide residues in length. However, the kinetic characteristics of the respective DNA interactions with 30S particles vary significantly. In addition to the investigation of normal 30S particles, a four base deletion within the 1400 region of 16S rRNA was analyzed. The deletion was made by using synthetic DNAs to target the deletion site for RNase H digestion. The direct in vitro procedure for manipulating rRNA conserves nucleotide modifications. The alteration causes a significant change in the disposition of 16S rRNA in 30S subunits, suggesting a reduction in the freedom of movement of the altered zone in the particle. In a factor-dependent in vitro protein synthesis system primed with MS2 mRNA and altered 30S subunits, there was a 50% decrease in phage coat protein synthesis. The reduction could be due to a decrease in the rate of translation or premature termination of translation. We present evidence here, based on isotopic studies, which supports the latter possibility.     

tRNA binding sites of ribosomes from Escherichia coli

70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites. The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding. It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM. A third binding site in addition to P and A sites was observed for deacylated tRNAPhe. At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low. Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites. These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome. Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely. Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one. This difference possibly indicates some specificity of the E. coli ribosome for tRNAs from the same organism.