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Biomedical subjects

S Altman

Publications and source records attributed to S Altman.

At least 127 records · Page 7Linked to original sources
PubMed1987

Heterologous enzyme function in Escherichia coli and the selection of genes encoding the catalytic RNA subunit of RNase P.

The gene for the catalytic RNA subunit of RNase P has been isolated from several Enterobacteriaceae by complementation of an Escherichia coli strain that is temperature-sensitive for RNase P activity. The selection procedure relies on the ability of the heterologous gene products to function enzymatically in E. coli. This procedure obviates the need for positive results in DNA blot hybridization experiments or for the purification of holoenzyme to identify the RNA component of RNase P and its corresponding gene from organisms other than E. coli. Comparisons of the variations in sequences provide the basis for a refined two-dimensional model of the secondary structure of M1 RNA.

Base Sequence↗
PubMed1986

Site-directed mutagenesis of M1 RNA, the RNA subunit of Escherichia coli ribonuclease P. The effects of an addition and small deletions on catalytic function.

One addition mutation and several small deletion mutations have been created in vitro at a unique site in the gene coding for M1 RNA, the RNA subunit of Escherichia coli RNase P. The mutant genes exhibit a wide range of efficiencies in complementing another mutant that is thermosensitive for RNase P function in vivo. The transcripts of the mutated genes cleave a precursor tRNA in vitro with efficiencies that parallel their ability to function in the complementation assay in vivo. The secondary structures in solution of the mutant gene transcripts are shown to be different from the parent molecule by probing the structure of the transcripts with ribonuclease T1. A local region of secondary structure, between nucleotides 275 and 295, must be maintained for normal function of M1 RNA.

Endoribonucleases↗
PubMed1986

M1 RNA with large terminal deletions retains its catalytic activity.

Truncated transcripts of the rnpB gene from E. coli, coding for M1 RNA, the catalytic subunit of RNAase P, and fragments of M1 RNA generated by nuclease treatment have been prepared, and their ability to function catalytically in vitro has been determined. Molecules missing as many as 122 nucleotides at the 3' terminus retain catalytic activity, although at a much lower level than M1 RNA itself. No activity is observed with an RNA that is missing 70 nucleotides at the 5' terminus. The removal of even a small number of nucleotides from both termini eliminates all catalytic function. The preservation of one intact terminus may be essential for the tertiary and quaternary interactions required to generate the conformation of an active RNA species.

Bacterial Proteins↗
PubMed1986

Metal ion requirements and other aspects of the reaction catalyzed by M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli.

M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli, can under certain conditions catalytically cleave precursors to tRNA in the absence of C5, the protein moiety of RNase P. M1 RNA itself is not cleaved during the reaction, nor does it form any covalent bonds with its substrate. Only magnesium and, to a lesser extent, manganese ions can function at the catalytic center of M1 RNA. Several other ions either inhibit the binding of magnesium ion at the active site or function as structural counterions. The reaction rate of cleavage of precursors to tRNAs by M1 RNA is enhanced in the presence of poly-(ethylene glycol) or 2-methyl-2,4-pentanediol. Many aspects of the reaction catalyzed by M1 RNA are compatible with a mechanism in which phosphodiester bond cleavage is mediated by metal ion.

Binding Sites↗
PubMed1986

M1 RNA, the RNA subunit of Escherichia coli ribonuclease P, can undergo a pH-sensitive conformational change.

After purification from extracts of whole cells, M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli, apparently must undergo a change in conformation before it can function catalytically. The rate of this conformational change is dependent upon the duration of incubation at various temperatures and pH. delta E of the transition at pH 7.5 is approximately 36 kcal/mol. The change in conformation is not sensitive to Mg2+ concentration between 10 and 100 mM. A decrease in A260 of M1 RNA in solution has been observed during the incubation period that potentiates the conformational change at 30 degrees C, but no direct correlation can yet be made to specific structural rearrangements.

Endoribonucleases↗
PubMed1986

Reconstitution of RNAase P activity using inactive subunits from E. coli and HeLa cells.

HeLa cell RNAase P activity found in the flow-through of anti-Sm affinity columns can be separated into inactive RNA and protein components. These components can be used to reconstitute active hybrid enzyme complexes with purified subunits from E. coli RNAase P. The RNA in the HeLa cell fractions employed is enriched for species between 85 and 115 nucleotides long. This reconstitution assay is a convenient means of purifying the functional RNA and protein of HeLa cell RNAase P. Probes derived from the genes for the subunits of E. coli RNAase P hybridize to genomic DNA of gram-negative prokaryotic organisms, but no positive signals are seen with genomic DNA from a variety of eukaryotic organisms.

Endoribonucleases↗
PubMed1986

Affinity chromatography with an immobilized RNA enzyme.

M1 RNA, the catalytic subunit of Escherichia coli RNase P, has been covalently linked at its 3' terminus to agarose beads. Unlike M1 RNA, which is active in solution in the absence of the protein component (C5) of RNase P, the RNA linked to the beads is active only in the presence of C5 protein. Affinity chromatography of crude extracts of E. coli on a column prepared from the beads to which the RNA has been crosslinked results in the purification of C5 protein in a single step. The protein has been purified in this manner from cells that contain a plasmid, pINIIIR20, which includes the gene that codes for C5 protein. A 6-fold amplification of the expression of C5 protein is found in these cells after induction as compared to cells that do not harbor the plasmid.

Chromatography, Affinity↗
PubMed1986

Different cation transport inhibitor in benign and malignant experimental renal hypertension.

The role of circulating humoral agents in the pathogenesis of abnormal vascular wall cation composition in benign and malignant renal hypertension was investigated. Male F344 rats with chronic benign (n = 38) and malignant (n = 44) one-kidney, one clip (1K1C) hypertension and normotensive control rats (n = 63) were studied. Malignant hypertension developed spontaneously and was characterized by failure to thrive, weight loss, oedema, renal insufficiency, anaemia or haemoconcentration and hyperkalaemia. For bio-assay, monolayers of quiescent vascular smooth muscle cells from F344 rats were incubated in plasma or plasma extracts of normotensive and hypertensive rats for measurement of labelled rubidium (86Rb) uptake in the presence and absence of 2 mmol/l ouabain and/or 1 mmol/l furosemide. Compared with controls, ouabain-sensitive Rb uptake of cells was reduced in plasma extracts but not in whole plasma of rats with benign hypertension. Ouabain-sensitive Rb uptake was unchanged and ouabain-insensitive Rb uptake was reduced in both plasma and plasma extracts of rats with malignant hypertension. The latter was due to a reduction in furosemide-sensitive Rb uptake. In malignant hypertension, the increased sodium (Na) content of the aorta which characterizes benign hypertension was reversed and bladder wall Na content was reduced. The findings suggest that in malignant hypertension a circulating, furosemide-like inhibitor of ouabain-insensitive cation transport is the cause of vascular wall Na depletion and of diuresis and natriuresis that trigger the syndrome.

Animals↗
PubMed1986

Cell growth and sodium content in the serum of uninephrectomized and renal hypertensive dogs.

The long-term cellular metabolic effects of serum factors from renal hypertensive and uninephrectomized normotensive dogs were investigated. Mouse fibroblastic L cells were cultured in M199 tissue culture medium supplemented (to 15%) with serum obtained from dogs before and after the induction of one-kidney one wrapped hypertension or with serum obtained from normotensive dogs before and after unilateral nephrectomy. At confluency, the protein, water (3-O-methyl-14C-D-glucose space), Na, K, and Mg content and 3H-thymidine uptake of cells were measured. Growth curves were derived for cells growing in serum obtained before and after uninephrectomy from normotensive and hypertensive dogs. Postnephrectomy serum from both normotensive and hypertensive dogs resulted in increased recovery of protein, increased water, K, and Mg content of cells, and increased cell numbers. 3H-thymidine uptake at confluency in postnephrectomy serum was the same as in prenephrectomy serum. The one important qualitative difference between postnephrectomy serum from normotensive and hypertensive dogs was the reduced Na content and concentration of cells cultured in the serum of hypertensive dogs. The growth-promoting serum factor in postnephrectomy blood was detected for up to 8 weeks after uninephrectomy; therefore, it did not appear to be renotropin. In experimental renal hypertension, there appear to be previously unidentified serum factors that enhance transmembrane Na gradient.

Animals↗
PubMed1985

Characterization of an RNase P activity from HeLa cell mitochondria. Comparison with the cytosol RNase P activity.

A ribonuclease P-like activity was partially purified from HeLa cell mitochondria by DEAE-cellulose and octyl-Sepharose chromatography. RNase P-like activity can be quantitatively recovered from intact mitochondrial preparations treated with micrococcal nuclease, strongly suggesting that the enzyme is localized within the organelles. Mitochondrial RNase P (mtRNase P) cleaves the precursor to Escherichia coli suppressor tRNATyr at the same site as E. coli RNase P, producing the mature 5'-end of tRNATyr. The sensitivity of mtRNase P to pretreatment with nucleases or Pronase indicates that the enzyme has essential RNA and protein components. Although the ionic requirements of mtRNase P are similar to those of the RNase P activity isolated from the post-mitochondrial cytosol fraction, the chromatographic properties of mtRNase P are distinct. Mitochondrial RNase P is probably a part of the mitochondrial RNA processing machinery of mammalian mitochondria, being responsible for the endonucleolytic cleavage of the RNA transcripts at the 5'-side of the tRNA sequences.

Adenosine Triphosphate↗
PubMed1985

A catalytic RNA and its gene from Salmonella typhimurium.

The gene for the RNA subunit (M1 RNA) of ribonuclease P from Salmonella typhimurium directs the synthesis of an RNA that can cleave transfer RNA precursor molecules. The mature M1 RNA coded for by Salmonella typhimurium is 375 nucleotides long and has six nucleotide changes in comparison to M1 RNA from Escherichia coli. The regions for promotion and termination of transcription are closely conserved, but adjacent regions of nucleotide sequences show considerable drift.

Base Sequence↗
PubMed1985

Novel non-suppressing mutants of Escherichia coli tRNATyr su+3.

Several addition and deletion mutations were constructed in the region of the gene for Escherichia coli tRNATyr su+3 corresponding to the dihydrouracil loop of the mature tRNA. None of these resulting mutants had detectable suppressor function compared to the parent gene yet some directed the synthesis of mature tRNA. These latter mutants may affect the ability of the tRNA to be aminoacylated or to interact with the translational machinery on the ribosome.

Chromosome Deletion↗
PubMed1984

Structure in solution of M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli.

The structure of M1 RNA, the RNA component of Escherichia coli RNase P, has been probed by mild digestion with a variety of ribonucleases. The results have been used to generate a model for the two-dimensional structure of M1 RNA. This model is similar in many respects to an earlier model that was based entirely on theoretical considerations. M1 RNA was digested with RNase T1 in buffer containing 10 mM MgCl2 (in which M1 RNA, by itself, has no catalytic activity) and in buffer containing 60 mM MgCl2 (in which M1 RNA can cleave precursors to tRNA molecules). Under these conditions, the main features of the secondary structure are similar, but several minor differences are apparent. Such subtle changes in structure are also observed when M1 RNA is present in a binary complex with a substrate molecule, the precursor to E. coli tRNATyr.

Endoribonucleases↗
PubMed1984

Catalytic activity of an RNA molecule prepared by transcription in vitro.

Ribonuclease P is a ribonucleoprotein that cleaves precursors to transfer RNA (tRNA) molecules to yield the correct 5' terminal sequences of the mature tRNA's. The RNA moiety M1 RNA of ribonuclease P from Escherichia coli and the unprocessed transcript prepared in vitro of the gene for M1 RNA can both perform the cleavage reactions of the canonical enzyme in the absence of the protein moiety. When the transcript of the M1 RNA gene is combined with the protein moiety not only is a tRNA precursor cleaved but also the precursor to 4.5S RNA from Escherichia coli.

Base Sequence↗
PubMed1984

Cleavage of tRNA precursors by the RNA subunit of E. coli ribonuclease P (M1 RNA) is influenced by 3'-proximal CCA in the substrates.

tRNA precursor molecules that contain the CCA sequence found at the 3' termini of all mature tRNAs are cleaved in vitro more readily by M1 RNA, the catalytic subunit of E. coli RNAase P, than precursors that lack this sequence. The sensitivity to the CCA sequence is not apparent when precursors are cleaved by the reconstituted RNAase P holoenzyme that contains both M1 RNA and the protein subunit. These results have been obtained with monomeric precursor molecules encoded by the E. coli and human chromosomes and with three dimeric precursor molecules encoded by the bacteriophage T4 genome. The data are in agreement with previous results concerning T4 tRNA biosynthesis in vivo and show that the CCA sequence is important for the processing of precursors to tRNAs.

Base Sequence↗