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Sidney Altman

Publications and source records attributed to Sidney Altman.

At least 19 recordsLinked to original sources
PubMed2005

Analysis of putative RNase P RNA from orthopoxviruses.

A putative RNase P RNA gene in camelpox virus, one of the orthopoxviruses, was cloned and transcribed in vitro. No RNase P activity could be detected in vitro from camelpox virus RNase P RNA alone, or by addition of the Escherichia coli RNase P protein subunit to reaction mixtures. Camelpox virus RNase P RNA reconstituted in vitro with camel or HeLa cell extracts, which were pre-treated with micrococcal nuclease to degrade the endogenous RNase P RNA, showed no RNase P activity. Vaccinia virus, another orthopoxvirus, showed no RNase P activity in vaccinia-infected HeLa cells, even though transcription of the vaccinia RNase P RNA could be identified in the cells by both Northern blot and RNase protection assay. Camelpox virus RNase P RNA inhibited an endogenous HeLa RNase P activity by 20% in our assays. The 5S RNA showed no significant inhibition in this assay.

Animals↗
PubMed2005

Regulated expression of functional external guide sequences in mammalian cells using a U6 RNA polymerase III promoter.

A regulatable promoter has been stably integrated into a human embryonic kidney cell line. The promoter is a pol III mouse promoter and is under the control of ponasterone A, an ecdysone inducer. The promoter controls transcription of an external guide sequence (EGS) targeted against Rpp38, a protein subunit of human RNase P, or of lamin A/C, a gene product located in the nucleus. The amounts of protein of both gene products are severely reduced when the EGSs are made. Several other, but not all, of the protein subunits of RNase P are also inhibited in both mRNA and protein levels when Rpp38 mRNA is targeted.

Animals↗
PubMed2005

RNase P cleaves transient structures in some riboswitches.

RNase P from Escherichia coli cleaves the coenzyme B12 riboswitch from E. coli and a similar one from Bacillus subtilis. The cleavage sites do not occur in any recognizable structure, as judged from theoretical schemes that have been drawn for these 5' UTRs. However, it is possible to draw a scheme that is a good representation of the E. coli cleavage site for RNase P and for the cleavage site in B. subtilis. These data indicate that transient structures are important in RNase P cleavage and in riboswitch function. Coenzyme B12 has a small inhibitory effect on E. coli RNase P cleavage of the E. coli riboswitch. Both E. coli RNase P and a partially purified RNase P from Aspergillus nidulans mycelia succeeded in cleaving a putative arginine riboswitch from A. nidulans. The cleavage site may be a representative of another model substrate for eukaryotic RNase P. This 5' UTR controls splicing of the arginase mRNA in A. nidulans. Four other riboswitches in E. coli were not cleaved by RNase P under the conditions tested.

Aspergillus nidulans↗
PubMed2005

Masters of DNA.

Explore the source record for details and available documents.

Animals↗
PubMed2004

Inhibition of the expression of the human RNase P protein subunits Rpp21, Rpp25, Rpp29 by external guide sequences (EGSs) and siRNA.

External guide sequences (EGSs) and siRNAs were targeted individually to the mRNA of three of the protein subunits of human RNase P, Rpp21, Rpp25 and Rpp29. The production of each of the three targets was inhibited in every specific case. In addition, some of the remaining protein subunits were also inhibited by these specific EGSs and the siRNAs. These data, in general, confirm previous results on the inhibition of a sub-group of all the protein subunits with an EGS against Rpp38. The effect of EGSs is apparent in 24 hours after transfection but the effect of siRNAs, which is comparable to the EGS data in amounts of inhibition, takes at least 48 to 96 hours to become evident. No general understanding of the mechanism of action of the siRNAs, in terms of which portion of a target mRNA they bind to for function, was apparent from the design of those used here.

Base Sequence↗
PubMed2004

In search of RNase P RNA from microbial genomes.

A simple procedure has been developed to quickly retrieve and validate the DNA sequence encoding the RNA subunit of ribonuclease P (RNase P RNA) from microbial genomes. RNase P RNA sequences were identified from 94% of bacterial and archaeal complete genomes where previously no RNase P RNA was annotated. A sequence was found in camelpox virus, highly conserved in all orthopoxviruses (including smallpox virus), which could fold into a putative RNase P RNA in terms of conserved primary features and secondary structure. New structure features of RNase P RNA that enable one to distinguish bacteria from archaea and eukarya were found. This RNA is yet another RNA that can be a molecular criterion to divide the living world into three domains (bacteria, archaea, and eukarya). The catalytic center of this RNA, and its detection from some environmental whole genome shotgun sequences, is also discussed.

Base Sequence↗
PubMed2004

Polarity effects in the lactose operon of Escherichia coli.

An intergenic RNA segment between lacY and lacA of the lactose operon in Escherichia coli is cleaved by RNase P, an endoribonuclease. The cleavage of the intergenic RNA was ten times less efficient than cleavage of a tRNA precursor in vitro. Fragments of the RNase P cleavage product are detectable in vivo in the wild-type strain but not in a mutant strain at the restrictive temperature. The cleavage product that contains lacA in the wild-type strain was quickly degraded. When this intergenic segment was cloned upstream of a reporter gene, the expression of the reporter gene was also inhibited substantially in wild-type E.coli, but not in a temperature sensitive mutant strain in RNase P at the restrictive temperature. These results support data regarding the natural polarity between lacZ versus lacA, the downstream gene.

Base Sequence↗
PubMed2004

Disruption of type III secretion in Salmonella enterica serovar Typhimurium by external guide sequences.

The type III secretion system involved in Salmonella enterica serovar Typhimurium invasion of host cells has been disrupted using inducibly expressed oligonucleotide external guide sequences (EGSs) complementary to invB or invC mRNA. These EGSs direct single site cleavage in these mRNAs by endogenous RNase P, and their expression in Salmonella results in invC mRNA and InvC protein depletion, decreased type III secretion and interference with host cell invasion. Comparison of these effects with those from studies of Salmonella invB and invC mutants suggests that invB EGSs have polar effects on invC mRNA.

Bacterial Proteins↗
PubMed2003

A specific endoribonuclease, RNase P, affects gene expression of polycistronic operon mRNAs.

The rnpA mutation, A49, in Escherichia coli reduces the level of RNase P at 43 degrees C because of a temperature-sensitive mutation in C5 protein, the protein subunit of the enzyme. Microarray analysis reveals the expression of several noncoding intergenic regions that are increased at 43 degrees C compared with 30 degrees C. These regions are substrates for RNase P, and they are cleaved less efficiently than, for example, tRNA precursors. An analysis of the tna, secG, rbs, and his operons, all of which contain RNase P cleavage sites, indicates that RNase P affects gene expression for regions downstream of its cleavage sites.

Base Sequence↗
PubMed2003

Coordinate inhibition of expression of several genes for protein subunits of human nuclear RNase P.

The deliberate inhibition of expression of one of the protein subunits (Rpp38) of human nuclear RNase P is achievable by using external guide sequence (EGS) technology. Both the protein product and the mRNA are greatly reduced 24 h after transient transfection with a gene coding for an appropriate EGS. Control experiments indicated that four other protein subunits of RNase P and their RNAs are also inhibited with no external manipulation. The remaining RNase P proteins, their mRNAs, and the RNA subunit of RNase P all are unchanged. Several short nucleotide sequences adjacent to the ORFs for the inhibited genes are similar and could be targets for transcriptional repression. The explanation of coordinate inhibition of the expression of the product of one particular gene by the transfection of an EGS (or RNA interference) requires some care in terms of interpreting phenotypic effects because, in our case, several gene products that are not targeted are also inhibited.

Base Sequence↗
PubMed2003

The effect of a single, temperature-sensitive mutation on global gene expression in Escherichia coli.

High-density DNA microarrays have been used to explore the genomic profiling of gene expression of a defective Escherichia coli strain with a temperature-sensitive mutation in the protein component of RNase P. A novel gene cluster was discovered in which two of the genes are known substrates of RNase P. The expression pattern of essential genes and gene discovery from intergenic regions, for which other new transcripts are found, are also discussed.

Bacterial Proteins↗
PubMed2002

Elucidation of structure-function relationships in the protein subunit of bacterial RNase P using a genetic complementation approach.

RNase P is a ribonucleoprotein involved in tRNA biosynthesis in all living organisms. Bacterial RNase P is comprised of a catalytic RNA subunit and a lone protein cofactor which plays a supporting, albeit essential, role in the tRNA processing reaction in vivo. In this study, we have searched various databases to identify homologs of the protein subunit of RNase P from diverse bacteria and used an alignment of their primary sequences to determine the most highly conserved residues, and thereby extend earlier predictions of which residues might play an important role in RNA recognition. By employing a genetic complementation assay, we have also gained insights into structure- function relationships in the protein subunit of bacterial RNase P.

Amino Acid Sequence↗
PubMed2002

Partial reconstitution of human RNase P in HeLa cells between its RNA subunit with an affinity tag and the intact protein components.

An RNA affinity tag was incorporated into the RNA subunit of human nuclear RNase P. The tagged RNA assembled with the protein components of RNase P inside HeLa cells to generate an active enzyme. Because of the specificity of the RNA tag to streptavidin, the reconstituted complex could be separated from the native enzyme and other ribonucleoproteins (particularly RNase MRP) by streptavidin agarose chromatography and could be recovered by the eluting agent, biotin. A mutant, tagged RNase P RNA, whose P3 domain was partially replaced, could not reconstitute with the proteins to yield an active enzyme. The P3 domain, therefore, is critical for the structure and function of RNase P.

Affinity Labels↗
PubMed2002

A protein subunit of human RNase P, Rpp14, and its interacting partner, OIP2, have 3'-->5' exoribonuclease activity.

The processing of precursor tRNAs at their 5' and 3' termini is a fundamental event in the biosynthesis of tRNA. RNase P is generally responsible for endonucleolytic removal of a leader sequence of precursor tRNA to generate the mature 5' terminus. However, much less is known about the RNase P counterparts or other proteins that are active at the tRNA 3' terminus. Here we show that one of the human RNase P subunits, Rpp14, together with one of its interacting protein partners, OIP2, is a 3'-->5' exoribonuclease with a phosphorolytic activity that processes the 3' terminus of precursor tRNA. Immunoprecipitates of a crude human RNase P complex can process both ends of precursor tRNA by hydrolysis, but purified RNase P has no exonuclease activity. Rpp14 and OIP2 may be part of an exosome activity.

Carrier Proteins↗
PubMed2002

Purification and characterization of Rpp25, an RNA-binding protein subunit of human ribonuclease P.

In HeLa cells, ribonuclease P (RNase P), the tRNA processing enzyme consists of an RNA subunit (H1 RNA) associated with at least nine protein subunits, Rpp14, Rpp20, Rpp21, Rpp29 (hPop4), Rpp30, Rpp38, Rpp40, hPop1, and hPop5 (18.8 kDa). We report here the cloning and immuno-biochemical analysis of Rpp25, another protein subunit of RNase P. Polyclonal rabbit antibodies raised against recombinant Rpp25 recognize their corresponding antigens in RNase P-containing fractions purified from HeLa cells, and they also precipitate active holoenzyme. Furthermore, this protein has general RNA binding properties.

Amino Acid Sequence↗