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At least 865 records · Page 48Linked to original sources
PubMed1988

Interaction of Escherichia coli ribosomal protein S8 with its binding sites in ribosomal RNA and messenger RNA.

The ability of ribosomal protein S8 from Escherichia coli to interact with 12 variants of its 16 S rRNA binding site, as well as with a regulatory sequence within spc operon mRNA, has been assessed. Single-site alterations were introduced into the appropriate segment of the E. coli 16 S rRNA gene by mutagenesis in vitro. Their effects on S8-rRNA interaction were measured via a filter-binding assay, utilizing S8 binding sites transcribed in vitro from the altered 16 S rRNA gene fragments. Of the 12 rRNA mutants, six were unable to bind S8. Significantly, five of these occur within a small, phylogenetically conserved internal loop, defined by nucleotides 596-597 and 641-643, suggesting that this structure plays a major role in S8-16 S rRNA recognition. The reduced affinity of S8 for its binding site in these cases was closely correlated with growth defects that resulted from expression of the same mutations in vivo. Alterations at other positions in the S8 binding site had little influence on complex formation or cell growth, as long as they did not disrupt rRNA secondary structure. The specific interaction of S8 with a segment of the spc operon mRNA containing a putative site of translational feedback regulation was demonstrated using appropriate in vitro transcripts in conjunction with the filter-binding assay. The apparent association constant for the S8-mRNA interaction was determined to be approximately 5 x 10(6) M-1, about five times lower than for the interaction of S8 with wild-type 16 S rRNA. The structure of the regulatory binding site, determined by sequence analysis of spc operon mRNA protected by S8 from RNase digestion, was found to contain all of the characteristic features of the 16 S rRNA binding site, demonstrating that the protein associates with structurally similar domains in both RNAs.

Base Sequence↗
PubMed2006

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata.

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC50 (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml(-1)) and by HeLa, HT29 and JM cells with IC50 in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml(-1), all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.

Amino Acid Sequence↗
PubMed1997

The ribosome-in-pieces: binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA.

An oligoribonucleotide (a 27-mer) that mimics the sarcin/ricin (S/R) domain of Escherichia coli 23S rRNA binds elongation factor EF-G; the Kd is 6.9 microM, whereas for binding to ribosomes it is 0.7 microM. Binding saturates when EF-G and the S/R RNA are equimolar; at saturation 70% of the input RNA is in complexes with EF-G. Binding of EF-G to S/R RNA does not require GTP but is inhibited by GDP; the inhibition by GDP is overcome by GTP. The effects of mutations of the S/R domain nucleotides G2655, A2660, and G2661 suggest that EF-G recognizes the conformation of the RNA rather than the identity of the nucleotides. EF-G also binds to an oligoribonucleotide (an 84-mer) that has the thiostrepton region of 23S rRNA; however, EF-G binds independently to S/R and thiostrepton oligoribonucleotides.

Base Sequence↗
PubMed1984

Structural and functional analysis of Escherichia coli ribosomes containing small deletions around position 1760 in the 23S ribosomal RNA.

Three different small deletions were produced at a single Pvu 2 restriction site in E. coli 23S rDNA of plasmid pKK 3535 using exonuclease Bal 31. The deletions were located around position 1760 in 23S rRNA and were characterized by DNA sequencing as well as by direct fingerprinting and S1-mapping of the rRNA. Two of the mutant plasmids, Pvu 2-32 and Pvu 2-33, greatly reduced the growth rate of transformed cells while the third mutant, Pvu 2-14 grew as fast as cells containing the wild-type plasmid pKK 3535. All three mutant 23S rRNAs were incorporated into 50S-like particles and were even found in 70S ribosomes and polysomes in vivo. The conformation of mutant 23S rRNA in 50S subunits was probed with a double-strand specific RNase from cobra venom. These analyses revealed changes in the accessibility of cleavage sites near the deletions around position 1760 and in the area around position 800 in all three mutant rRNAs. We suggest, that an altered conformation of the rRNAs at the site of the deletion is responsible for the slow growth of cells containing mutant plasmids Pvu 2-32 and Pvu 2-33.

Base Sequence↗
PubMed1984

Diversity of the ribosomal structures in the Euglena gracilis chloroplast genome: description of a mutant with two ribosomal operons and possible mechanism for its production.

We studied the bleached mutant of Euglena gracilis Y3BUD producing carotenoids and rich in chloroplast DNA. This mutant which was produced more than twenty years ago segregates permanently, forming 30 to 50% daughter cells without carotenoids and poor in chloroplast DNA. Electron microscopy and mapping with restriction enzymes show the deletion of a complete ribosomal operon out of the three present in the original strain.

Chloroplasts↗
PubMed1986

Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli.

Twelve specific alterations have been introduced into the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA. Appropriate rDNA segments were first cloned into bacteriophage M13 vectors and subjected to bisulfite and oligonucleotide-directed mutagenesis in vitro. Subsequently, the mutagenized sequences were placed within the rrnB operon of plasmid pNO1301 and the mutant plasmids were used to transform E. coli recipients. The growth rates of cells containing the mutant plasmids were determined and compared with that of cells containing the wild-type plasmid. Only those mutations which occurred at highly conserved positions, or were expected to disrupt the secondary structure of the binding site, increased the doubling time appreciably. The most striking changes in growth rate resulted from mutations that altered a small internal loop within the S8 binding site. This structure is phylogenetically conserved in prokaryotic 16S rRNAs and may play a direct role in S8-16S rRNA recognition and interaction.

Base Sequence↗
PubMed1979

Fluorescence studies of the accessibility of the 3' ends of the ribosomal RNAs in Escherichia coli ribosomes and subunits.

The accessibility of the 3'-ends of E. coli in various states has been probed by reaction, after periodate oxidation, with the fluorescent dye proflavine semicarbazide. Free oxidized 16S and 23S rRNAs each react with 2 equivalents of dye. The 23S rRNA is equally reactive in the 50S subunit and the 70S ribosome. The 16S RRNA 3'-end is accessible in the 30S subunit. In the intact 70S particle, periodate can reach the 3'-end of the 16S rRNA but the dye cannot. The 5S rRNA is relatively inaccessible to periodate oxidation or dye reaction in the 70S particle. Dye-labelled 16S rRNA will reconstitute into 30S particles but they are inactive in polypeptide synthesis. This is apparently due to the inability of the 30S particles to form tight complexes with 50S subunits. Iodide quenching studies indicate that the environment of the 3'-end of 16S rRNA in the 30S particle is different from that of the free rRNA.

Escherichia coli↗
PubMed1978

Polynucleotide-protein interactions in the translation system. Detection of contacts between some ribosomal split proteins and 16-S RNA in 30-S subunits of Escherichia coli ribosomes.

Direct contacts between 16-S RNA and split proteins S2, S3, S5, S14 and S21 inside the 30-S subunit of Escherichia coli ribosomes were evidenced by the formation of ultraviolet-induced (lambda = 254 nm) RNA-protein cross-links. 30-S subunits were reassembled from core particles and a mixture of split proteins containing in each case a single 125I-labelled protein. All the proteins tested are cross-linked as a result of a single-hit process; proteins S3 and S21 were cross-linked at the highest rate.

Dose-Response Relationship, Radiation↗
PubMed1983

Ribosomal proteins cross-linked to 28-S and 18-S rRNA separated by sedimentation after ultraviolet irradiation of rat-liver ribosomes.

Rat liver 80-S ribosomes, irradiated with 254 nm ultraviolet light for 15-60 min (0.63-2.5 X 10(5) quanta/ribosome), were treated with sodium dodecyl sulfate; 18-S and 28-S RNA fractions were isolated by sucrose density-gradient centrifugation. The protein moieties of the 18-S and 28-S rRNA fractions were labeled with 125I. After digestion of the rRNA fractions with RNase A and T1, the cross-linked proteins were analyzed by two-dimensional acrylamide gel electrophoresis and then autoradiography. Cross-linked proteins were examined further by dodecyl sulfate/acrylamide gel electrophoresis of individual radioactive spots of proteins and their proteolytic peptides on two-dimensional gels. The results showed that S2 and S15 were cross-linked to 18-S rRNA, and L5, L6 and L8 to 28-S rRNA. Similar results were obtained when isolated 40-S or 60-S subunits were irradiated.

Animals↗
PubMedDate not supplied

[Possibility of internal organization of RNA and proteins in ribosomal subparticles. A structural model of Escherichia coli 30S ribosomal subparticle].

The hypothetic model of reciprocal spatial arrangement of 18 from 21 proteins in the E. coli 30S ribosomal subparticle is suggested. The model is based on conception of the 16S R-A molecule macrostrand which is the right superhelix in the subparticle composition. Macrohelix's biopolarity against single-stranded sites of RNA and its small width result in that proteins binding with single-stranded RNA organized in chain, one-number sequence. The double helixes uniting the corresponding one single-stranded sites of RNA play the role of rigid transmission between them. So, in the course of subparticles reconstruction from RNA and proteins the spatially uncoupled proteins can interact without its direct contact. The model takes into consideration the vast amount of information.

Binding Sites↗
PubMed1988

Immunological homologies between yeast ribosomal protein L2 and rat liver ribosomal proteins L4 and L24.

Polyclonal antibodies raised against ribosomal protein (r-protein) L2 of Schizosaccharomyces pombe were used to check for cross-reactions with total r-proteins of rat liver. Using this procedure, the rat liver r-proteins, L4 and L24, were identified as being immunologically related to yeast L2. In additional, homologies between rat liver L4 and L24 were detected. The possible implications for the regulation of r-protein synthesis are discussed.

Animals↗
PubMed1977

Phosphorylation of acidic ribosomal proteins from rabbit reticulocytes by a ribosome-associated casein kinase.

Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate polyacrylamide gel analysis of the purified acidic proteins and 80-S particles showed identical phosphoproteins in the 16 000 dalton region.

Adenosine Triphosphate↗
PubMed1982

Structural domains of ribosomal protein S8 and their relationship to ribosomal RNA binding.

Escherichia coli ribosomal protein S8 has been subjected to mild proteolytic digestion in order to search for structural domains within the protein [1]. A characteristic fragment produced in high yield after chymotrypsin treatment has been located with the protein sequence. Circular dichroism has shown this domain to be rich in alpha helix. However, the fragment loses its ability to bind to 16S rRNA as does a similar fragment produced by trypsin cleavage. The intact protein is required for rRNA binding and is highly protected against proteolytic digestion when bound to the RNA.

Amino Acid Sequence↗
PubMed1984

Hybrid 5 S ribosomal RNA encoded by a multicopy plasmid is incorporated into ribosomes of Escherichia coli.

The recombinant plasmid pJR3 delta contains a tandem pair of promoters from rrnA followed by a hybrid 5 S rRNA gene, derived from the two 5 S rRNA genes of the rrnD transcription unit, and a terminator. Escherichia coli cells transformed with this plasmid produce 2-3-times more 5 S rRNA compared to untransformed cells. The growth of cells containing this plasmid is not affected significantly. Although the sequence and the secondary structure of the plasmid-specific 5 S rRNA differ from those of its counterparts (e.g., from 5 S rRNA species encoded by chromosomal genes), it is processed properly and is incorporated into ribosomes.

Base Sequence↗
PubMed1981

Growth-rate-dependent regulation of ribosome synthesis in E. coli: expression of the lacZ and galK genes fused to ribosomal promoters.

Hybrid transducing phages were constructed in vitro that carry the galK gene fused to each of three ribosomal promoters: the promotor for an rRNA operon (rrnE); the promoter for the spec r protein operon and the promotor for the alpha r protein operon. We also constructed hybrid transducing phages that carry the IacZ gene fused to the promoter for the rrnE operon or to the promoter for the spc r protein operon. The amounts of galactokinase (or beta-galactosidase) were analyzed in lysogens carrying these various transducing phages grown in several different growth media. The synthesis rate of galactokinase (or beta-galactosidase) from the fused rrn-gal (or rrn-lac) operon relative to the total protein synthesis rate increased with increasing growth rate, as expected from the transcriptional activity of rRNA operons. In contrast, the relative synthesis rate of galactokinase (or beta-galactosidase) from the operon fused to alpha or spc r protein promoter remained approximately constant with increasing growth rate. These results were interpreted to mean that the characteristic increase in the relative synthesis rate of r protein with increasing growth rate is determined not by transcription regulatory mechanisms, but by posttranscriptional mechanisms, which presumably involve the feedback inhibition of r protein mRNA translation by free r proteins.

Escherichia coli↗
PubMed1994

Proteins of the Thermus thermophilus ribosome. Purification of proteins from the large ribosomal subunit.

Special procedures have been developed to isolate and purify 26 of the 30 individual proteins of the large ribosomal subunit from Thermus thermophilus. Sixteen of them have been purified under non-denaturing conditions to be used for crystallization and further structural studies. These proteins have been characterized by their amino acid content, molecular mass, UV-spectrum and extinction coefficient. An additional 10 proteins have been purified by reverse phase chromatography. Thirteen proteins have been identified by homological E coli proteins.

Amino Acid Sequence↗
PubMed2005

A determination of the identity elements in yeast 18 S ribosomal RNA for the recognition of ribosomal protein YS11: the role of the kink-turn motif in helix 11.

A description of the site of interaction of YS11, the yeast homolog of eubacterial S17, with 18 S rRNA was obtained by assessing the binding of the ribosomal protein, in a filter retention assay, to oligoribonucleotides that reproduce regions of 18 S rRNA. YS11 binds predominantly to domain I; the Kd value is 113 nM. The dimensions of the YS11 binding site were refined, guided by chemical protection data and by the atomic structure of the Thermus thermophilus 30 S subunit, which has the S17 recognition site in 16 S rRNA. An oligoribonucleotide that mimics helix 11, a phylogenetically conserved region in domain I, binds YS11 with a Kd value of 230 nM; a second oligoribonucleotide that contains only the kink-turn motif in helix 11 binds YS11 with a Kd value of 528 nM. Thus, helix 11 has most of the nucleotides required for the recognition of YS11. To identify those nucleotides a set of 27 transversion mutations in H11 was constructed and their contribution to the binding of YS11 determined. Mutations of nine nucleotides (U313, C314, A316, G337, C338, G347, U348, U350, and C351) increased the Kd value for YS11 binding by at least eightfold; G325U and U349A mutations increased the Kd value fivefold. Eight of the 11 mutations are in the kink-turn in H11, confirming the critical importance of the motif for YS11 recognition. The other three nucleotides are in the lower stem and the terminal loop of H11, which makes a lesser, but still important, contribution to YS11 binding. The identity elements for YS11 recognition are: A316, G325, G337, G347, U348, U349, U350, and C351. The effect of the other nucleotides that decrease binding is probably indirect, presumably they affect the conformation of the binding site but do not have contacts to YS11 amino acid residues. The eight identity element nucleotides are in regions of H11 that deviate from A-form geometry and the contacts are predominantly, if not exclusively, to backbone phosphate and sugar oxygen atoms, indicating that YS11 recognizes the shape of the rRNA binding site rather than reading the sequence of nucleotides.

Amino Acid Sequence↗
PubMed1998

Identification by site-directed mutagenesis of amino acid residues in ribosomal protein L2 that are essential for binding to 23S ribosomal RNA.

The ribosomal protein L2 (BstL2) from Bacillus stearothermophilus is a primary 23S rRNA binding protein. We made use of site-directed mutagenesis to identify essential basic and aromatic amino acid residues for 23S rRNA binding. Four mutants, R68Q, K70Q, R86Q, and R155Q, in which Arg-68, Lys-70, Arg-86, and Arg-155, respectively, are replaced by the Gln residue. showed reduced binding affinities as compared with that of the wild type BstL2 (a binding constant K=8.93 microM(-1)): K values of these mutants range between 0.24 and 1.86 microM(-1). As for aromatic amino acids, replacements of Phe-66, Tyr-95 or Tyr-102 by alanine significantly abolished the binding affinities. CD analysis of the mutant proteins indicated that the mutations of four basic residues (Arg-68, Lys-70, Arg-86 and Arg-155) did not affect protein structure, whereas those of aromatic residues (Phe-66, Tyr-95, and Tyr-102) appeared to cause slight structural perturbations. These results, together with sequence comparison of L2 family proteins, suggest that Arg-86 and Arg-155 in BstL2 may act as positively charged recognition groups for negatively charged phosphate backbone of the 23S rRNA, and that Phe-66, Tyr-95, and Tyr-102 may be candidate residues which stabilize the BstL2-23S rRNA interaction through intramolecular interactions.

Alanine↗