Biomedical subjects

I G Wool

Publications and source records attributed to I G Wool.

At least 19 recordsLinked to original sources

PubMed2000

The phenotype of mutations of the base-pair C2658.G2663 that closes the tetraloop in the sarcin/ricin domain of Escherichia coli 23 S ribosomal RNA.

The sarcin/ricin domain (SRD) in Escherichia coli 23 S rRNA is a part of the site for the association of elongation factors with ribosomes and for that reason is critical for the binding of aminoacyl-tRNA and for translocation during the reiterative elongation reactions of protein synthesis. The SRD has a GAGA tetraloop that is shut off by a Watson-Crick C2658 x G2663 pair. The contribution of this pair to the function of the ribosome has been evaluated by constructing mutations in the nucleotides and determining their phenotype. Constitutive expression of a plasmid-encoded rrnB operon with a G2663C transversion mutation that disrupts the Watson-Crick pair was lethal. Double transversion mutations, C2658G x G2663C and C2658A x G2663U, that reverse the polarity of the pyrimidine and the purine but restore the potential to form a canonical pair, were also lethal. Induction of transcription of 23 S rRNA with the same mutations, but encoded in a plasmid with a lambdaP(L) promoter and expressed at a lower level, retarded growth. The sedimentation profiles of ribosomes with transversion mutations in C2658 and/or G2663 are altered; the ratio of 50 S subunits to 30 S particles is changed and polysomes are reduced. Ribosomes with a G2663C, a C2658G x G2663C, or a C2658A x G2663U mutation in 23 S rRNA were not active in protein synthesis, indeed, they appeared to inhibit the activity of ribosomes with wild-type 23 S rRNA. Transversion mutations in the analogs of C2658 and G2663 decreased binding of EF-G to SRD oligoribonucleotides; the same mutations in 23 S rRNA decreased binding of the factor to intact ribosomes. The most severe phenotype, in growth, in protein synthesis, and in the binding of EF-G, was associated with a C2658G x G2663C mutation; it is surprising that this was more severe than an analogous C2658A x G2663U mutation. A double transition mutation, C2658U x G2663A, which is not known to have occurred in nature, had no effect on the growth of cells or on the function of ribosomes. The lethal phenotype of transversion mutations in C2658 and G2663 appears to derive from a loss of the capacity of ribosomes to bind EF-G and by indirection the EF-Tu ternary complex.

Bacterial Proteins↗

PubMed1999

The contribution of a zinc finger motif to the function of yeast ribosomal protein YL37a.

Eukaryotic ribosomes have a large number of proteins but the exact nature of their contribution to the structure and to the function of the particle is not known. Of the 78 proteins in yeast ribosomes, six have zinc finger motifs of the C2-C2 variety. Both genes encoding the essential yeast ribosomal protein YL37a, which has such a zinc finger motif, were disrupteXXPd. The double deletion, which is lethal, can be rescued with a plasmid-encoded copy of a YL37a gene. Mutations were constructed in a plasmid-encoded copy of YL37a; the mutations caused the cysteine residues in the motif (at positions 39, 42, 57 and 60) to be replaced, one at a time, with serine. The cysteine residue at position 39, the first of the four in the motif, is essential for the function of YL37a, since a C39S mutation did not complement the null phenotype. However, plasmids encoding variants with C42S, C57S, or C60S mutations in the zinc finger motif were able to rescue the null mutant. YL37a binds zinc, but none of the mutant proteins, C39S, C42S, C57S, or C60S, was able to bind the metal. Thus, all four cysteine residues are essential for the binding of zinc; only one, C39, is essential for the function of the ribosomal protein.

Amino Acid Sequence↗

PubMed1999

The two faces of the Escherichia coli 23 S rRNA sarcin/ricin domain: the structure at 1.11 A resolution.

The sarcin/ricin domain of 23 S - 28 S ribosomal RNA is essential for protein synthesis because it forms a critical part of the binding site for elongation factors. A crystal structure of an RNA of 27 nucleotides that mimics the domain in Escherichia coli 23 S rRNA was determined at 1.11 A resolution. The domain folds into a hairpin distorted by four non-canonical base-pairs and one base triple. The fold is stabilized by cross-strand and intra-stand stacking; no intramolecular stabilizing metal ions are observed. This is the first structure to reveal in great detail the geometry and the hydration of two common motifs that are conserved in this rRNA domain, a GAGA tetraloop and a G-bulged cross-strand A stack. Differences in the region connecting these motifs to the stem in the E. coli and in the rat sarcin/ricin domains may contribute to the species-specific binding of elongation factors. Correlation of nucleotide protection data with the structure indicates that the domain has two surfaces. One surface is accessible, lies primarily in the major groove, and is likely to bind the elongation factors. The second lies primarily in the minor groove, and is likely to be buried in the ribosome. This minor groove surface includes the Watson-Crick faces of the cytosine bases in the unusual A2654.C2666 and U2653.C2667 water-mediated base-pairs.

Animals↗

PubMed1999

The phenotype of mutations of G2655 in the sarcin/ricin domain of 23 S ribosomal RNA.

The sarcin/ricin domain (SRD) in Escherichia coli 23 S rRNA forms a part of the site for the association of the elongation factors with the ribosome and hence is critical for the binding of aminoacyl-tRNA and for translocation. The domain is also the site of action of the eponymous toxins which catalyze covalent modification of single nucleotides that inactivate the ribosome. The conformation of the conserved guanosine at position 2655 is an especially prominent feature of the structure of the SRD: the nucleotide is bulged out of a helix and forms a base-triple with A2665 and U2656. G2655 in 23 S rRNA is protected from chemical modification when the elongation factors, EF-Tu and EF-G, are bound to ribosomes and the analog of G2655 in oligoribonucleotides is critical for recognition by the toxin sarcin and by EF-G. The contribution of G2655 to the function of the ribosome has been evaluated by constructing mutations in the nucleotide and determining the phenotype. Constitutive expression of a plasmid-encoded rrnB operon with a deletion of, or transversions in, G2655 is lethal to E. coli cells, whereas a defect in the growth of cells with a G2655A transition is observed only in competition with wild-type cells. The sedimentation profiles of ribosomes with mutations in G2655 are altered; most markedly by deletion or transversion of the nucleotide, less severely by transition to adenosine. Mutations of G2655 confer resistance to sarcin on ribosomes. Ribosomes with G2655Delta, G2655C, or G2655U mutations in 23 S rRNA are not active in protein synthesis, whereas those with the G2655A transition mutation suffer decreased activity.

Base Sequence↗

PubMed1999

Characterization of in vitro and in vivo mutations in non-conserved nucleotides in the ribosomal RNA recognition domain for the ribotoxins ricin and sarcin and the translation elongation factors.

The sarcin/ricin domain in 23 S/28 S rRNA is crucial for ribosome function, since it constitutes at least part of the binding site for the elongation factors and hence is essential for binding aminoacyl-tRNA and for translocation. The domain is also the site of action of ricin and sarcin and analysis of the effect of mutations in the RNA on recognition by the cytotoxins has helped to define the structure and to understand the function of the region. We have constructed deletions, separately, of pairs of non-conserved, juxtaposed but non-hydrogen-bonded nucleotides that correspond to C4317 and C4331, and to U4316 and C4332, in an oligoribonucleotide that mimics the sarcin/ricin domain in rat 28 S rRNA. The deletions had no effect on the depurination of A4324 by ricin nor on the cleavage of the phosphodiester bond on the 3' side of G4325 by sarcin. However, simultaneous deletion of the four nucleotides decreased cleavage by sarcin but did not affect depurination by ricin. Removal of the non-canonical A4318.A4330 pair abolished recognition by both toxins. Deletion from oligoribonucleotides, that reproduce the sarcin/ricin domain of Escherichia coli 23 S rRNA, of U2653 and C2667 (equivalent to U4316, C4317 and C4331, C4332 in 28 S rRNA), or substitution of guanosine for U2653 (designed to form a Watson-Crick G2653.C2667 pair), reduced cleavage by sarcin whereas depurination by ricin was slightly increased. An increase in the stability of the mutant oligoribonucleotides may be the basis of the impairment in sarcin action. The tm for the wild-type RNA is 60 degreesC; for the double-deletion mutant U2653Delta/C2667Delta it is 65 degreesC; and for the U2653G transversion it is 69 degreesC. Expression of a mutant 23 S rRNA gene lacking U2653 and C2667 is lethal and a U2653G transversion mutation impairs growth. The mutant ribosomes are less active in protein synthesis than the wild-type and ribosomes with the U2653G mutation are resistant to sarcin. The binding of EF-G to oligoribonucleotides with a U2653/C2667 double deletion is reduced and an effect on the affinity of the factor for the sarcin/ricin domain may account in part for the decrease in ribosome efficiency. The results stress the potential importance in rRNA structure and function of non-conserved nucleotides, and suggest that the sarcin/ricin domain in ribosomes requires a region of structural flexibility for optimal efficiency.

Animals↗

PubMed1998

Crystal structure of the ribosomal RNA domain essential for binding elongation factors.

The structure of a 29-nucleotide RNA containing the sarcin/ricin loop (SRL) of rat 28 S rRNA has been determined at 2.1 A resolution. Recognition of the SRL by elongation factors and by the ribotoxins, sarcin and ricin, requires a nearly universal dodecamer sequence that folds into a G-bulged cross-strand A stack and a GAGA tetraloop. The juxtaposition of these two motifs forms a distorted hairpin structure that allows direct recognition of bases in both grooves as well as recognition of nonhelical backbone geometry and two 5'-unstacked purines. Comparisons with other RNA crystal structures establish the cross-strand A stack and the GNRA tetraloop as defined and modular RNA structural elements. The conserved region at the top is connected to the base of the domain by a region presumed to be flexible because of the sparsity of stabilizing contacts. Although the conformation of the SRL RNA previously determined by NMR spectroscopy is similar to the structure determined by x-ray crystallography, significant differences are observed in the "flexible" region and to a lesser extent in the G-bulged cross-strand A stack.

Animals↗

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↗

PubMed1996

The primary structures of rat ribosomal proteins S3a (the V-Fos transformation effector) and of S3b.

The amino acid sequence of the rat 40S ribosomal subunit protein S3a was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed by the determination of the NH2-terminal sequence by Edman degradation. Ribosomal protein S3a has 263 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and the molecular weight is 29,794. The protein designated S3b has the same amino acid sequence as S3a except that it lacks the carboxyl-terminal 12 residues. We are unable to determine whether there are separate genes for S3a and S3b, or whether there is a single gene and alternate splicing of the precursor to yield separate mRNAs for S3a and S3b, or whether there is a single gene and a single mRNA whose translation yields S3a which is converted by proteolysis, either physiological or fortuitous, to S3b. The mRNA for S3a is about 1000 nucleotides in length. Hybridization of cDNA to digests of nuclear DNA suggests that there are 8-13 copies of the S3a gene. Rat ribosomal protein S3a is identical to the product of the rat Fte-1 gene which encodes the V-Fos transformation effector; S3a is also related to the plant protein cyc07, which is encoded by a cell cycle S-phase specific gene.

Alternative Splicing↗

PubMed1996

The primary structure of rat ribosomal protein L10: relationship to a Jun-binding protein and to a putative Wilms' tumor suppressor.

The amino acid sequence of the rat 60S ribosomal subunit protein L10 was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed by determination of the NH2-terminal amino acid sequence in the protein. Ribosomal protein L10 has 213 amino acids (the NH2-terminal methionine is removed after translation of the mRNA); the molecular weight is 24,456. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 8 to 10 copies of the L10 gene. The mRNA for the protein is about 900 nucleotides in length. Rat L10 is related to ribosomal proteins from other eukaryotes. Ribosomal protein L10 is, in addition, the mammalian homolog of the chicken Jun-binding protein and is nearly identical to a putative Wilms' tumor suppressor. This is a presumptive example, of which there are many others, of an extraribosomal function of a ribosomal protein.

Amino Acid Sequence↗

PubMed1996

The primary structure of rat ribosomal protein L14.

The amino acid sequence of the rat 60S ribosomal subunit protein L14 was deduced from the sequence of nucleotides in two recombinant cDNAs. Ribosomal protein L14 has 213 amino acids (the NH2-terminal methionine is removed after translation of the mRNA); the molecular weight is 23,193. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 6 to 8 copies of the L14 gene. The mRNA for the protein is about 800 nucleotides in length. Rat L14 is related to a number of previously unidentified ribosomal proteins from other eukaryotes but not to any from archaebacteria or eubacteria. The carboxyl-terminal amino acid sequences of rat L14 and of chicken histone H1 are related. Mutation of the Drosophila melanogaster homolog of rat L14 causes a severe Minute phenotype.

Amino Acid Sequence↗

PubMed1996

The primary structure of rat ribosomal protein L10a.

The amino acid sequence of the rat 60S ribosomal subunit protein L1Oa was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L10a has 217 amino acids and has a molecular weight of 24,815. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7 to 1O copies of the L10a gene. The mRNA for the protein is about 760 nucleotides in length. Rat L1Oa is related to ribosomal proteins from other eukaryotes and from archaebacteria.

Amino Acid Sequence↗

PubMed1996

Determination of the 28 S ribosomal RNA identity element (G4319) for alpha-sarcin and the relationship of recognition to the selection of the catalytic site.

Ricin A-chin and alpha-sarcin are ribotoxins that inactivate eukaryotic ribosomes by modifying 28 S rRNA; ricin A-chain is an RNA N-glycosidase that depurinates the adenosine at position 4324 and alpha-sarcin is a ribonuclease that cleaves the phosphodiester bond on the 3' side of the adjacent guanosine (at position 4325). In cartoons of the secondary structure these two residues are seen to be embedded in a 17 base single-stranded loop over a seven base-pair helix. However, NMR spectroscopy of an oligoribonucleotide, a 29-mer that mimics the sarcin/ricin domain, indicates that the RNA has a compact conformation in which the guanosine at the position analogous to 4319 in 28 S rRNA is bulged out of what otherwise is an extended A-form helix. Since similar structural irregularities are used by proteins to bind to RNA, we have tested the effect of mutations of the bulged guanosine on recognition and covalent modification of the RNA by ricin A-chain and by alpha-sacrin. For the test a synthetic oligoribonucletide, a 35-mer, was used; the mutations were the deletion, the transition to adenosine, and the transversion to cytidine and uridine of the guanosine that is the analog of G4319. Each of the four mutations abolished cleavage og the RNA by alpha-sacrin, where depurination by ricin A-chain was little affected. Thus G4319 is an identity element for alpha-sacrin recognition. Analysis of the effect of alpha-sacrin on variant oligoribonucleotides in which additional bases were inserted between the identity element guanosine and the site of catalysis suggest that on binding to the RNA the toxin uses the guanosine for orientation and then cleaves at a fixed distance and at a fixed position in space.

Base Sequence↗

PubMed1996

Dependence of depurination of oligoribonucleotides by ricin A-chain on divalent cations and chelating agents.

Ricin A-chain is a cytotoxic RNA N-glycosidase that inactivates eukaryotic ribosomes by depurinating the adenosine at position 4324 in 28S rRNA. The enzyme retains its specificity when a synthetic oligoribonucleotide (a 35-mer) that mimics the structure at the site of action is the substrate. However, covalent modification by ricin A-chain of the oligoribonucleotide but not of ribosomes, depends on the simultaneous presence of a divalent cation and a chelating agent.

Base Sequence↗

PubMed1996

The primary structure of rat ribosomal protein L6.

The amino acid sequence of the rat 60S ribosomal subunit protein L6 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed by determination of amino acid sequences in the protein. Ribosomal protein L6 has 296 amino acids (the NH2-terminal methionine is removed after translation of mRNA); the molecular weight is 33,417. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7 to 10 copies of the L6 gene. The mRNA for the protein is about 1,100 nucleotides in length. Rat L6 is related to ribosomal proteins from other eukaryotes and to histone H1.

Amino Acid Sequence↗

PubMed1996

Extraribosomal functions of ribosomal proteins.

The discovery of DNA-binding motifs in ribosomal proteins has led to the conjecture that the transition of the ribosome from an RNA to an RNP machine occurred by adding pre-existing proteins. Supportive, but circumstantial, evidence for the hypothesis is adduced from the finding that many ribosomal proteins have a second function apart from the particle. These extraribosomal functions are enumerated.

Biological Evolution↗

PubMed1995

Systematic deletion analysis of ricin A-chain function. Single amino acid deletions.

The A-chain of ricin is a cytotoxic RNA N-glycosidase that inactivates ribosomes by depurination of the adenosine at position 4324 in 28 S rRNA. Of the 267 amino acids in the protein, 222 could be deleted, in one or another of 74 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA (Morris, K. N., and Wool, I. G. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4869-4873). The 45 amino acids that could not be omitted when the deletions were in sets of 20, 5, or 2 residues have now been deleted one at a time; 9 of these deletion mutants retained activity. A RNP-like structural motif in ricin A-chain that may mediate binding to ribosomal RNA has been identified.

Amino Acid Sequence↗

PubMed1995

The carboxyl extensions of two rat ubiquitin fusion proteins are ribosomal proteins S27a and L40.

Two rat recombinant cDNAs were characterized; they encode fusion proteins that have at their NH2 terminus the conserved 76 amino acid ubiquitin and at their carboxyl terminus the extension ribosomal proteins S27a (80 amino acids and a molecular weight of 9,397) or L40 (52 amino acids and a molecular weight of 6,177). The fusion proteins are processed in a reticulocyte lysate to ubiquitin and either S27a or L40. Hybridization of cDNAs to digests of nuclear DNA suggests that there are 14 to 19 copies of the S27a, and 6 to 10 of the L40, genes. The mRNA for ubiquitin-S27a has about 700 nucleotides and ubiquitin-L40 about 650. Ribosomal proteins S27a and L40 contain zinc finger motifs of the C2-C2 variety. S27a and L40 are related to ribosomal proteins from other species.

Amino Acid Sequence↗

PubMed1995

The primary structures of rat ribosomal proteins L4 and L41.

The amino acid sequences of the rat 60S ribosomal subunit proteins L4 and L41 were deduced from the sequences of nucleotides in recombinant cDNAs. Ribosomal protein L4 has 421 amino acids; the molecular weight is 47,280. L41 is the smallest ribosomal protein; it has 25 amino acids and a molecular weight of 3,454. Hybridization of the cDNAs to digests of nuclear DNA suggests that there are 7 to 8 copies of the L4, and 9 to 12 of the L41, genes. The mRNA for L4 is about 1,500 nucleotides in length and that for L41 about 500 nucleotides. The 5' noncoding sequence of the L4 cDNA is exceptional in that it has, in addition to a short polypyrimidine sequence at the 5' end, a second stretch of 15 consecutive pyrimidines near the site of initiation of translation. The 3' noncoding sequence of the L41 mRNA is unusual in that it is at least 246 nucleotides long. Rat L4 and L41 are related to ribosomal proteins from other eukaryotes.

Amino Acid Sequence↗