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S Altman

Publications and source records attributed to S Altman.

At least 109 records · Page 6Linked to original sources
PubMed1990

Protection from chemical modification of nucleotides in complexes of M1 RNA, the catalytic subunit of RNase P from E coli, and tRNA precursors.

Certain nucleotides in M1 RNA, the catalytic RNA subunit of RNase P from E coli, are protected from chemical modification when M1 RNA forms complexes with tRNA precursor molecules (ES complexes). Many of these nucleotides are important in the formation of the Michaelis complex. In the presence of tRNA precursor molecules, the pattern of protection from chemical modification of a region in M1 RNA that resembles the E site in 23S rRNA is similar to the pattern of protection of the E site in the presence of deacylated tRNA. In the complex with the RNA enzyme, more nucleotides in the substrate become accessible to modification, an indication that the substrate is in an unfolded conformation under these conditions.

Base Sequence↗
PubMed1989

Specific interactions in RNA enzyme-substrate complexes.

Analysis of crosslinked complexes of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli, and transfer RNA precursor substrates has led to the identification of regions in the enzyme and in the substrate that are in close physical proximity to each other. The nucleotide in M1 RNA, residue C92, which participates in a crosslink with the substrate was deleted and the resulting mutant M1 RNA was shown to cleave substrates lacking the 3' terminal CCAUCA sequence at sites several nucleotides away from the normal site of cleavage. The presence or absence of the 3' terminal CCAUCA sequence in transfer RNA precursor substrates markedly affects the way in which these substrates interact with the catalytic RNA in the enzyme-substrate complex. The contacts between wild-type M1 RNA and its substrate are in a region that resembles part of the transfer RNA "E" (exit) site in 23S ribosomal RNA. These data demonstrate that in RNA's with very different cellular functions, there are domains with similar structural and functional properties and that there is a nucleotide in M1 RNA that affects the site of cleavage by the enzyme.

Base Sequence↗
PubMed1989

Catalysis by the RNA subunit of RNase P--a minireview.

RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5' termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate.

Catalysis↗
PubMed1989

Reaction in vitro of some mutants of RNase P with wild-type and temperature-sensitive substrates.

The reaction of wild-type and two mutant derivatives of RNase P have been examined with wild-type and mutant substrates. We show that a mutant derivative of tRNA(Tyr)Su3, tRNA(Tyr)Su3A15, in which the G15.C48(57) base-pair essential for folding of the tRNA moiety is altered, is a temperature-sensitive suppressor in vivo. The precursor to tRNA(Tyr)Su3A15 is cleaved in a temperature-sensitive manner in vitro by RNase P and with a higher Km compared to the precursor to tRNA(Tyr)Su3. The precursor to tRNA(Tyr)Su3A2, another temperature-sensitive suppressor in vivo in which the G2.C71(80) base-pair in the acceptor stem is changed to A2.C71(80), behaves like the precursor to tRNA(Tyr)Su3 in vitro; that is, it is not cleaved in a temperature-sensitive manner. Therefore, there are at least two ways in which a suppressor tRNA can acquire a temperature-sensitive phenotype in vivo. One of the mutant derivatives of RNase P we have tested, rnpA49, which affects the protein cofactor of the enzyme, has a decreased kcat compared to wild-type, which can explain its phenotype in vivo.

Endoribonucleases↗
PubMed1989

An immunological determinant of RNase P protein is conserved between Escherichia coli and humans.

RNase P, an enzyme with RNA and protein subunits, cleaves tRNA precursor molecules to form the 5' termini of mature tRNAs in both prokaryotes and eukaryotes. Rabbit antibodies made against the protein subunit, C5 protein, of Escherichia coli RNase P bound RNase P protein from E. coli and Bacillus subtilis in immunoblots and solid-phase immunoassays. These rabbit anti-C5 antibodies also bound a protein (Mr approximately 40,000) in preparations of RNase P from human (HeLa) cells and depleted the enzymatic activity from preparations of RNase P from both human and E. coli cells. Finally, rabbit anti-C5 antibodies immunoprecipitated from crude extracts of human cells a ribonucleoprotein complex containing H1 RNA, the putative RNA component of human RNase P. These results show that an antigenic determinant is shared by C5 protein from E. coli RNase P and a protein component of RNase P from human cells.

Antibodies↗
PubMed1989

Identification and characterization of an RNA molecule that copurifies with RNase P activity from HeLa cells.

An RNA molecule, 340 nucleotides in length and designated H1 RNA, copurifies with RNase P activity from extracts of HeLa cells or isolated HeLa cell nuclei. When the genomic DNA of various organisms is probed with H1 cDNA in Southern hybridization assays, only mammalian DNA gives a positive signal. The gene coding for H1 RNA in human cells is present in one to three copies per cell. The nucleotide sequence of H1 RNA, which shows little homology to the known sequences of its analogs from prokaryotes and yeast, can be drawn as a two-dimensional, hydrogen-bonded structure that resembles similar structures proposed for the RNA subunit of RNase P from these other sources. Part of the hypothetical structure is virtually identical to structures that can be drawn for analogous RNAs from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and S. octosporus.

Blotting, Southern↗
PubMed1989

Characterization in vitro of the defect in a temperature-sensitive mutant of the protein subunit of RNase P from Escherichia coli.

We have studied the assembly of Escherichia coli RNase P from its catalytic RNA subunit (M1 RNA) and its protein subunit (C5 protein). A mutant form of the protein subunit, C5A49, has been purified to apparent homogeneity from a strain of E. coli carrying a thermosensitive mutation in the rnpA gene. The heat inactivation kinetics of both wild-type and mutant holoenzymes are similar, an indication of equivalent thermal stability. However, when the catalytic efficiencies of the holoenzymes were compared, we found that the holoenzyme containing the mutant protein had a lower efficiency of cleavage than the wild-type holoenzyme at 33, 37, and 44 degrees C. We then explored the interaction of M1 RNA and C5 protein during the assembly of the holoenzyme. The yield of active holoenzyme obtained by reconstitution with wild-type M1 RNA and C5A49 protein in vitro can be considerably enhanced by the addition of excess M1 RNA, just as it can be in vivo. We concluded that the Arg-46----His-46 mutation in the C5A49 protein affects the ability of the protein to participate with M1 RNA in the normal assembly process of RNase P.

Endoribonucleases↗
PubMed1988

Differential effects of mutations in the protein and RNA moieties of RNase P on the efficiency of suppression by various tRNA suppressors.

We have studied the efficiency of suppression by tRNA suppressors in vivo in strains of Escherichia coli that harbor a mutation in the rnpA gene, the gene for the protein component (C5) of RNase P, and in strains that carry several different alleles of the rnpB gene, the gene for the RNA component (M1) of RNase P. Depending on the genetic background, different efficiencies of suppression by the various tRNA suppressors were observed. Thus, mutations in rnpA have separable and distinct effects from mutations in rnpB on the processing of tRNA precursors by RNase P. In addition, the efficiency of suppression by several derivatives of E. coli tRNA(Tyr) Su3 changed as the genetic background was altered.

Bacterial Proteins↗
PubMed1988

Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli.

The genes for the protein (C5 protein) and RNA (M1 RNA) subunits of Escherichia coli RNase P have been subcloned and their products prepared in milligram quantities by rapid procedures. The interactions between the two subunits of the enzyme have been studied in vitro by a filter-binding technique. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant for the specific interactions of the subunits in the holoenzyme complex is approximately 4 x 10(-10) M. C5 protein also interacts with various RNA molecules in a non-specific manner with a dissociation constant of 2 x 10(-8) to 6 x 10(-8) M. Regions of M1 RNA required for interaction with C5 protein have been defined by deletion analysis and footprinting techniques. These interactions are localized primarily between nucleotides 82 to 96 and 170 to 270 of M1 RNA.

Bacterial Proteins↗
PubMed1988

Selection and characterization of randomly produced mutants in the gene coding for M1 RNA.

The gene for M1 RNA, the catalytic subunit of RNase P of Escherichia coli, was subjected to random chemical mutagenesis in vitro. Mutations were selected by electrophoresis in denaturing gradient gels. Twenty-seven different mutants of the gene for M1 RNA were selected, and in 24 cases the mutations were identified as single base substitutions. The mutant forms of M1 RNA were analyzed in vitro for catalytic activity in the absence and in the presence of the protein subunit of RNase P (C5 protein). The structure of mutant RNAs was probed by limited digestion with ribonuclease T1; a correlation between reduced catalytic activity of mutant M1 RNAs and perturbations in secondary and tertiary structure was noted in many cases. The results indicate the involvement of specific regions of the M1 RNA molecule in the catalytic function of RNase P, in the binding of the C5 protein, and in substrate binding.

Endoribonucleases↗
PubMed1988

Novel reactions of RNAase P with a tRNA-like structure in turnip yellow mosaic virus RNA.

A quasi-continuous double hellix, containing a pseudoknot and ending in a single-stranded region which contains CCA, can be formed at the 3' terminus of the genomic RNAs of certain plant viruses. M1 RNA (the catalytic subunit) alone and the RNAase P holoenzyme from E. coli cleave the tRNA-like structure of TYMV RNA in vitro at the 5' side of the quasi-helical structure to generate 5' phosphate and 3' hydroxyl groups in the cleavage products. The intact pseudoknot structure in the substrate is not required for the reaction catalyzed by M1 RNA alone, but its presence markedly improves the efficiency of the reaction. In addition to its essential role in the biosynthesis of tRNA, RNAase P may have another function in vivo, namely, in the physiology of viral infections.

Base Sequence↗
PubMed1988

The recognition by RNase P of precursor tRNAs.

We have generated mutants of M1 RNA, the catalytic subunit of Escherichia coli RNaseP, and have analyzed their properties in vitro and in vivo. The mutations, A333----C333, A334----U334, and A333 A334----C333 U334 are within the sequence UGAAU which is complementary to the GT psi CR sequence found in loop IV of all E. coli tRNAs. We have examined: 1) whether the mutant M1 RNAs are active in processing wild type tRNA precursors and 2) whether they can restore the processing defect in mutant tRNA precursors with changes within the GT psi CR sequence. As substrates for in vitro studies we used wild type E. coli SuIII tRNA(Tyr) precursor, and pTyrA54, a mutant tRNA precursor with a base change that could potentially complement the U334 mutation in M1 RNA. The C333 mutation had no effect on activity of M1 RNA on wild type pTyr. The U334 mutant M1 RNA, on the other hand, had a much lower activity on wild type pTyr. However, use of pTyrA54 as substrate instead of wild type pTyr did not restore the activity of the U334 mutant M1 RNA. These results suggest that interactions via base pairing between nucleotides 331-335 of M1 RNA and the GT psi CG of pTyr are probably not essential for cleavage of these tRNA precursors by M1 RNA. For assays of in vivo function, we examined the ability of mutant M1 RNAs to complement a ts mutation in the protein component of RNaseP in FS101, a recA- derivative of E. coli strain A49. In contrast to wild type M1 RNA, which complements the ts mutation when it is overproduced, neither the C333 nor the U334 mutant M1 RNAs was able to do so.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Antibodies in human serum that precipitate ribonuclease P.

Sera from certain patients with systemic lupus erythematosus (SLE) and related rheumatic diseases contain antibodies that selectively deplete extracts of HeLa cells of RNase P activity. Most of the sera that recognize RNase P, an endoribonuclease with an essential RNA subunit, also contain antibodies against another small ribonucleoprotein known as the Th antigen. A species of RNA about 400 nucleotides in length is the only RNA species found in common in all immunoprecipitates prepared with anti-RNase P antibodies. The discovery of antibodies against RNase P defines a major class of antibodies produced by patients with autoimmune disease.

Autoantibodies↗
PubMed1987

Model substrates for an RNA enzyme.

M1 RNA, the catalytic RNA subunit of Escherichia coli ribonuclease P, can cleave novel transfer RNA (tRNA) precursors that lack specific domains of the normal tRNA sequence. The smallest tRNA precursor that was cleaved efficiently retained only the domain of the amino acid acceptor stem and the T stem and loop. The importance of the 3' terminal CCA nucleotide residues in the processing of both novel and normal tRNA precursors implies that the same enzymatic function of M1 RNA is involved.

Base Sequence↗
PubMed1987

Relation of serum total cholesterol and high-density lipoprotein cholesterol percentage to the incidence of definite coronary events: twenty-year follow-up of the Donolo-Tel Aviv Prospective Coronary Artery Disease Study.

This 20-year follow-up report presents the incidence of definite coronary events and its relation to serum total cholesterol (TC) and alpha-lipoprotein cholesterol percentage (percentage of serum TC bound with alpha-lipoprotein), now usually called high-density lipoprotein cholesterol percentage (HDL-C%). The cohort consists of 1,454 men and 1,481 women, presumably healthy, aged 35 to 64 years at the time of entrance examination in 1964. During the follow-up period 123 men and 44 women died from coronary disease. Ninety-six men and 42 women had nonfatal myocardial infarctions. The incidence of definite coronary events increased from 6% in men with TC levels of less than 200 mg/dl to 25% in men with levels of more than 264 mg/dl. The corresponding figures in women were 3% and 10%. The incidence of definite coronary events was inversely correlated to HDL-C%. The incidence of definite coronary events in 225 men with HDL-C% of less than 14% was 28%, and in 669 men with HDL-C% of 21% or more, 7%. The corresponding figures in 234 women with HDL-C% of less than 17% was 14%, and in 853 women with HDL-C% of 23% or more, 2%. Two HDL-C% subsets were assessed: "low" and "high" subsets refer to HDL-C% of less than 21% and 21% or more, respectively, in men and less than 23% and 23% or more in women.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Role in translation of a triple tandemly repeated sequence in the 5'-untranslated region of human thymidylate synthase mRNA.

A triple tandem repeat (TTR) consisting of 90 nucleotides exists immediately upstream of the ATG initiator codon in human thymidylate synthase (TS) cDNA (pcHTS-1). To investigate the role of the TTR in the expression of the TS cDNA, we used pcHTS-1 to construct mutant cDNA clones in which part of the TTR was deleted or an additional element was inserted. The mutant cDNA plasmid was introduced into murine TS-negative mutant cells and the relative translation efficiencies of the mutant cDNAs were determined by measuring the transient expression of TS activity and the amount of TS mRNA transcribed. The translation efficiency in transient expression of the mutants was increased by deletions covering all the first two repeated elements, and the part of the third closest to the ATG initiator codon, but was not affected by deletions of only parts of the first two repeated elements at the 5' end. The translation efficiency was also not affected by insertion of an additional repeated element into the TTR. These results suggest that the first two repeated elements at the 5' end both have inhibitory effects on translation of the TS mRNA, probably due to the unique structural feature of this element.

Animals↗
PubMed1987

Suppressor and novel mutants of bacteriophage T4 tRNA(Gly).

We have isolated a weak UGA suppressor of phage T4 tRNA(Gly) in which the anticodon is changed from UCC to UCA. Two secondary mutants lacking suppressor activity are atypical in accumulating tRNA(Gly). Both mutations change the T stem of the cloverleaf model. One involved a G to A change at the 5' base position of the middle base-pair; the second involves a C to U change at a constant base position next to the T loop. The precursor RNAs of the mutants were cleaved in vitro with the catalytic RNA subunit of RNase P. Relative to normal precursor RNA, the precursor mutated at the middle base-pair position of the T stem was cleaved more rapidly, whereas the precursor mutated at the base-pair position next to the T loop was cleaved more slowly.

Base Sequence↗