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Biomedical subjects

R A Garrett

Publications and source records attributed to R A Garrett.

At least 91 records · Page 5Linked to original sources
PubMed1985

Does unpaired adenosine-66 from helix II of Escherichia coli 5S RNA bind to protein L18?

Adenosine-66 is unpaired within helix II of Escherichia coli 5S RNA and lies in the binding site of ribosomal protein L18. It has been proposed as a recognition site for protein L18. We have investigated further the structural importance of this nucleotide by deleting it. The 5S RNA gene of the rrnB operon of E. coli was subjected to primer-directed mutagenesis. To produce the deletion it was necessary to use simultaneously the mutagenic dodecamer dCGGCGCACGGCG and the universal M13 primer dCCCAGTCACGACGTT, and to employ forced annealing conditions. The mutated gene was expressed in an overproducing plasmid derived from pKK3535. Binding studies with protein L18 revealed that the protein bound much more weakly to the mutated 5S RNA. We consider the most likely explanation of this result is that L18 interacts with adenosine-66, and we present a tentative model for an interaction between the unpaired adenosine and the adjacent guanosine-67 of the RNA and glutamine-19 of the protein L18.

Adenosine↗
PubMed1984

Higher-order structure in the 3'-terminal domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus.

An experimental approach was used to determine, and compare, the higher-order structure within domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus. This domain, which encompasses approximately 300 nucleotides at the 3' end of the RNAs, consists of two large subdomains. The 5' subdomain has been conserved during evolution and appears to be functionally important for the binding of the EF-1 X GTP X aminoacyl-tRNA complex in eukaryotes. The 3' subdomain has diverged widely between eubacteria and eukaryotes, and has produced the 4.5 S RNA in the chloroplast ribosomes of flowering plants. The structure of domain VI within the eubacterial RNAs was probed with chemical reagents in order to establish the degree of stacking and/or accessibility of each adenosine, cytidine and guanosine residue; the double-helical segments were localized with the cobra venom ribonuclease from Naja naja oxiana, and the relatively unstructured and accessible sequences were detected with the single-strand-specific ribonucleases A, T1 and T2. The data enabled the three secondary structural models, proposed for the E. coli 23 S RNAs, to be examined critically and it was concluded that many of their structural features are correct. Various differences between the models were considered and evidence is provided for additional structuring in the RNA including the stacking of juxtaposed purines into double helices. The 5' subdomain constitutes a compact and resistant structure whereas the 3' subdomain is relatively accessible and contains most of the potential protein binding sites. Moreover, comparison of our results with the published results on 4.5 S RNA suggests that the latter forms essentially the same structure as the 3' subdomain, in contrast to earlier conclusions. A high level of structural conservation has occurred throughout the RNA domain during the evolution of the Gram negative and Gram positive bacteria although the thermophile was generally more stable at base-pairs adjacent to the terminal loops.

Alkylating Agents↗
PubMed1984

Structure of a protein L23-RNA complex located at the A-site domain of the ribosomal peptidyl transferase centre.

Protein L23 from the ribosome of Escherichia coli is the primary ribosomal product cross-linked to affinity-labelled puromycin; it lies, therefore, within the A-site domain of the peptidyl transferase centre on the 50 S subunit. We have characterized this functional domain by isolating and sequencing the RNA binding site of protein L23; it consists of two main fragments of 25 and 105 nucleotides that strongly interact and are separated by 172 nucleotides in the primary sequence. The higher-order structure of the RNA moiety was probed by chemical reagents, and by single-strand and double-strand-specific ribonucleases; a secondary structural model and a tertiary structural interaction are proposed on the basis of these data that are compatible with phylogenetic sequence comparisons. Several nucleotides exhibited altered chemical reactivity, both lower and higher, in the presence of protein L23, thereby implicating a large proportion of the RNA structure in the protein binding. The sites were located mainly at the extremities of the helices and at nucleotides that were putatively bulged out from the helices. The RNA moiety and an adjacent excised fragment contain several highly conserved sequences and a modified adenosine. Such sequences constitute important functional domains of the RNA and may contribute to the putative role of this RNA region in the peptidyl transferase centre.

Acyltransferases↗
PubMed1984

The nucleotide sequence of the 16S ribosomal RNA gene of the archaebacterium Halococcus morrhua.

The sequence of the 16S rRNA gene from the archaebacterium Halococcus morrhua was determined by the dideoxynucleotide sequencing method. It is 1475 nucleotides long. This is the second archaebacterial sequence to be determined and it provides sequence comparison evidence for the secondary structural elements confined to the RNAs of this kingdom and, also, support for controversial or additional base pairing in the eubacterial RNAs. Six structural features are localized that have varied during the evolution of the archaebacteria, eubacteria and eukaryotes. Moreover, although the secondary structures of both sequenced archaebacterial RNAs strongly resemble those of eubacteria, they contain sufficient eukaryotic-like structural characteristics to reinforce the view that they belong to a separate line of evolutionary descent.

Archaea↗
PubMed1984

Retroperitoneal fibrosis in children (eosinophilic and idiopathic).

Two cases of retroperitoneal fibrosis in children are presented; one was idiopathic while the other was in association with eosinophilic cystitis, heretofore not reported. A literature search of patients under eighteen years of age reviews presentation, treatment, and associated diseases.

Child↗
PubMed1983

Higher order structure in the 3'-minor domain of small subunit ribosomal RNAs from a gram negative bacterium, a gram positive bacterium and a eukaryote.

An experimental approach was used to determine and compare the highest order structure within the 150 to 200 nucleotides at the 3'-ends of the RNAs from the small ribosomal subunits of Escherichia coli, Bacillus stearothermophilus and Saccharomyces cerevisiae. Chemical reagents were employed to establish the degree of stacking and/or accessibility of each adenosine, guanosine and cytidine. The double helices were probed with a cobra venom ribonuclease from Naja naja oxiana, and the relatively unstructured and accessible sequences were localized with the single strand-specific ribonucleases A, T1, T2 and S1. The data enabled the various minimal secondary structural models, proposed for the 3'-regions of the E. coli and S. cerevisiae RNAs, to be critically examined, and to demonstrate that the main common features of these models are correct. The results also reveal the presence and position of additional higher order structure in the renatured free RNA. It can be concluded that a high level of conservation of higher order structure has occurred during the evolution of the gram negative and gram positive eubacteria and the eukaryote in both the double helical regions and the "unstructured" regions. Several unusual structural features were detected. Multiple G X A pairings in two of the putative helices, which are compatible with phylogenetic sequence comparisons, are strongly supported by the occurrence of cobra venom ribonuclease cuts adjacent to, and in one case between, these pairings. Evidence is also provided for the stacking of an A X A pair within a double helix of the yeast RNA. Other special structural features include adenosines bulged out from double helices; such nucleotides, which are hyper-reactive, have been implicated in protein recognition in 5 S ribosomal RNA. The 3'-terminal regions of the RNAs are particularly important for the functioning of the ribosome. They are involved in mRNA, tRNA and ribosomal factor binding. The results reveal that while the functionally important RNA sequences tend to be conserved, they are not always accessible in the free RNA; the pyrimidine-rich "Shine and Dalgarno" sequence, for example, which is involved in mRNA recognition, occurs in a double helix in both eubacterial RNAs.

Base Sequence↗
PubMed1983

Comparison of Escherichia coli tRNAPhe in the free state, in the ternary complex and in the ribosomal A and P sites by chemical probing.

tRNAPheE.coli was modified at accessible guanosine, cytidine, and adenosine residues using the chemical modification method described by Peattie and Gilbert [Proc. Natl Acad. Sci. USA, 77, 4679-4689 (1980)]. Modification characteristics of the tRNA in the free state, in the ternary complex with elongation factor EF-Tu and GTP and in the ribosomal A and P sites were compared. A special procedure was devised to monitor, exclusively, tRNA molecules in the aminoacylated state. In the free tRNA, the most reactive bases are confined to the A73-C-C-A sequence of the aminoacyl stem, the anticodon loop, the D-loop and the extra loop and the results correlate well with the three-dimensional structure of tRNAPheyeast determined by X-ray studies. The pattern of reactivity was not affected either by charging the tRNA with phenylalanine or by labelling the 3' terminus with pCp. In the ternary complex, with elongation factor EF-Tu and GTP, changes in modification were observed at two sites, A73-C-C-A at the 3' terminus and C-13 and C-17 in the D-loop region, which are about 6 nm apart; no difference was observed in the anticodon loop. tRNAPhe bound at the ribosomal A or P sites exhibited similar, but not identical, modification patterns. Whereas nucleotides C-74 and C-75 were strongly protected at both sites, the adjacent A-73 showed an enhanced reactivity in the A site. The anticodon region G34-A-A-ms2.6(1)A was also strongly protected at both sites. In addition, nucleotide A-21 was protected during A-site, but not P-site, binding.

Binding Sites↗
PubMed1983

Specificity of anti-nucleoside antibodies in systemic lupus erythematosus.

The titer of IgG antinucleoside antibodies in the sera of 162 individuals was determined by an enzyme-linked immunosorbent assay. The nucleosides used in the assay were adenosine, cytidine, guanosine, and thymine-riboside conjugated to human serum albumin. The specificity of IgG antinucleoside antibodies was indicated by appropriate reduction in antibody binding after solid-phase adsorptions of antibody with specific immobilized nucleoside conjugates. Disease-associated increases in serum IgG antibodies to cytidine and guanosine but not to adenosine or thymine-riboside occurred in patients with systemic lupus erythematosus (SLE). The epitope density of nucleosides in the conjugates and differences in the sensitivity of each nucleoside assay were not responsible for disease-associated IgG antinucleoside antibody responses. These findings support a possible pathogenic role for cytidine and guanosine as antigens or crossreactive antigenic determinants in some patients with SLE.

Adenosine↗
PubMed1982

Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases.

The binding sites of ribosomal proteins L18 and L25 on 5S RNA from Escherichia coli were probed with ribonucleases A, T1, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S., & Garrett, R. A. (1981) Biochemistry 20, 7301--7307], reveal an extensive interaction site for protein L18 and a more localized one for L25. Generally comparable results, with a few important differences, were obtained in a study of the binding sites of the two E. coli proteins on Bacillus stearothermophilus 5S RNA. Several protein-induced changes in the RNA structures were identified; some are possibly allosteric in nature. The two prokaryotic 5S RNAs were also incubated with total 50S subunit proteins from E. coli and B. stearothermophilus ribosomes. Homologous and heterologous reconstitution experiments were performed for both RNAs. The effects of the bound proteins on the ribonuclease digestion of the RNAs could generally be correlated with the results obtained with the E. coli proteins L18 and L25, although there was evidence for an additional protein-induced conformational change in the B. stearothermophilus 5S RNA, which may have been due to a third ribosomal protein L5.

Bacterial Proteins↗
PubMed1981

Secondary structure of prokaryotic 5S ribosomal ribonucleic acids: a study with ribonucleases.

The structures of 5S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus were examined by using ribonucleases A, T1, and T2 and a double helix specific cobra venom ribonuclease. By using both 5' and 3'-32P-end labeling methods and selecting for digested but intact 5S RNA molecules, we were able to distinguish between primary and secondary cutting positions and also to establish the relative degree of cutting. The data reveal the predicted similarities of the higher order structure in the two RNAs but also demonstrate a few significant differences. The data also provide direct evidence for three of the helical regions of the Fox and Woese model of 5S RNA [Fox, G. E., & Woese, C. (1975) Nature (London) 256, 505] and support other important structural features which include a nucleotide looped out from a helical region which has been proposed as a recognition site for protein L18.

Base Sequence↗
PubMed1981

A "bulged" double helix in a RNA-protein contact site.

The binding of ribosomal protein L18 affects specific nucleotides in Escherichia coli 5S RNA as detected by dimethyl sulfate alkylation and RNase A digestion of the 5S-L18 complex. Most of the affected nucleotides are clustered and localize a site of RNA-protein interaction in and around the defined central helix [Fox, G. E. & Woese, C. (1975) Nature (London) 256, 505-507] of 5S RNA. Chemical carbethoxylation of the native 5S RNA with diethyl pyrocarbonate shows that a striking feature of this region is an unstacked adenosine residue at position 66. We propose that this residue exists as a singly bulged nucleotide extending the Fox and Woese central helix by two base pairs in the E. coli sequence (to positions 16-23/60-68) as well as in each of 61 (prokaryotic and eukaryotic) aligned 5S RNA sequences. In each case, the single bulged nucleotide is at the relative position of adenosine-66 in the RNA sequences. The presence of this putative bulged nucleotide appears to have been conserved in 5S RNA sequences throughout evolution, and its identity varies with major phylogenetic divisions. This residue is likely involved in specific 5S RNA-protein recognition or interaction in prokaryotic and eukaryotic ribosomes. The uridine-65 to adenosine-66 internucleotide bond is protected from RNase A digestion in the complex, and carbethoxylation of E. coli adenosine-66 prior to L18 binding affects formation of a stable RNA-protein complex. Thus, we identify a region of E. coli 5S RNA protected by the ribosomal protein L18 and propose that it contains a bulged nucleotide residue important in stable formation of this RNA-protein complex. This bulged residue appears to be evolutionarily conserved and phylogenetically defined in 5S RNA sequences in general, and consideration of other known RNA-protein binding sites shows that such a "bulged helix" may be a common feature of RNA-protein contact sites.

Base Sequence↗
PubMed1980

The role of the basic N-terminal region of protein L18 in 5S RNA-23S RNA complex formation.

Of the three proteins, L5, L18 and L25, which bind to 5S RNA, the former two effect the interaction of 5S RNA with 23S RNA. We have used trypsin as a probe to investigate the roles of the proteins in this RNA-RNA assembly, with the following results: (1) In complexes with 5S RNA, the highly basic N-terminal region of L18 is accessible to trypsin. This accessibility is unaffected by L25. However, its presence is essential for stimulating L5 binding. (2) In 5S RNA-protein-23S RNA complexes proteins L5 and L18 are both strongly resistant to proteolysis. (3) No 5S RNA-23S RNA complex formation occurs in the presence of L5 and the C-terminal L18 fragment. Two possible models for the mechanism of RNA-RNA assembly are proposed.

Bacterial Proteins↗
PubMed1980

Perirenal urinary extravasation associated with urethral valves in infants.

We present 7 cases of urethral valves and perirenal urinary extravasation, and review 37 cases reported previously in the literature. The extravasation usually is from the kidney (66 per cent) and often is associated with reflux (70 per cent). It appears to be a pressure-related phenomenon. Although the mortality associated with the condition is significant (32 per cent) renal function is usually preserved. Prompt treatment with correction of metabolic acidosis, and decompression of the tense abdomen and the urinary tract is recommended.

Ascites↗
PubMed1980

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.

Base Sequence↗