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J Abelson

Publications and source records attributed to J Abelson.

At least 109 records · Page 6Linked to original sources
PubMed1986

Mutational analysis of the coordinate expression of the yeast tRNAArg-tRNAAsp gene tandem.

tRNA genes occur in the yeast genome as highly dispersed and independent transcriptional units. The 5'-tRNAArg-tRNAAsp-3' gene tandem, separated by a 10-base-pair spacer sequence, thus represents a rare case of tight clustering. Previous in vitro studies did not reveal any primary transcript from the tRNAAsp gene, but rather a dimeric precursor containing both gene sequences plus spacer, which undergoes a series of maturation steps. This seems anomalous since the tRNAAsp gene contains the sequences necessary for its own transcription. We found that site-directed mutation of the highly conserved C at position 56 to a G in the tRNAArg gene suppresses all transcription and does not activate the tRNAAsp gene. Precise deletion of the entire tRNAArg gene gives a similar result. Rescue of tRNAAsp gene transcription is effected either by the precise deletion of both the tRNAArg gene and spacer or by the precise deletion of this gene with concomitant introduction of an artificial RNA polymerase III start site in the spacer. This artificial start site is ineffective if the tRNAArg gene is present upstream.

Base Sequence↗
PubMed1986

Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo.

The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.

In Vitro Techniques↗
PubMed1986

Intron mutations affect splicing of Saccharomyces cerevisiae SUP53 precursor tRNA.

The Saccharomyces cerevisiae amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the role of intron structure and sequence on precursor tRNA splicing in vivo and in vitro. This gene encodes a pre-tRNA which contains a 32-base intervening sequence. Two types of SUP53 intron mutants were constructed: ones with an internal deletion of the natural SUP53 intron and ones with a novel intron. These mutant genes were transcribed in vitro, and the end-processed transcripts were analyzed for their ability to serve as substrates for the partially purified S. cerevisiae tRNA endonuclease and ligase. The in vitro phenotype of these mutant RNAs was correlated with the in vivo suppressor tRNA function of these SUP53 alleles after integration of the genes into the yeast genome. Analysis of these mutant pre-tRNAs, which exhibited no perturbation of the mature domain, clearly showed that intron structure and sequence can have profound effects on pre-tRNA splicing. All of the mutant RNAs, which were inefficiently spliced or unspliced, evidenced cleavage only at the 5' splice junction. Base changes in the intron proximal to the 3' splice junction could partially rescue the splicing defect. The implications of these data for tRNA endonuclease-substrate interactions are discussed.

Computer Simulation↗
PubMed1985

Yeast mRNA splicing in vitro.

Synthetic actin and CYH2 pre-mRNAs containing a single intron are accurately spliced in a soluble whole cell extract of yeast. Splicing in vitro requires ATP. The excised intron is released as a lariat in which an RNA branch connects the 5' end of the molecule to the last A in the "intron conserved sequence" UACUAAC. Two other discrete RNA species produced during splicing in vitro may represent reaction intermediates: free, linear exon 1 and a form of the intron lariat extending beyond the 3' splice site to include exon 2. Both lariat forms correspond to molecules previously shown to be produced during yeast pre-mRNA splicing in vivo.

Actins↗
PubMed1985

Sequence organization and control of transcription in the bacteriophage T4 tRNA region.

Bacteriophage T4 contains genes for eight transfer RNAs and two stable RNAs of unknown function. These are found in two clusters at 70 X 10(3) base-pairs on the T4 genetic map. To understand the control of transcription in this region we have completed the sequencing of 5000 base-pairs in this region. The sequence contains a part of gene 3, gene 1, gene 57, internal protein I, the tRNA genes and five open reading frames which most likely code for heretofore unidentified proteins. We have used subclones of the region to investigate the kinetics of transcription in vivo. The results show that transcription in this region consists of overlapping early, middle and late transcripts. Transcription is directed from two early promoters, one or two middle promoters and perhaps two late promoters. This region contains all of the features that are seen in T4 transcription and as such is a good place to study the phenomenon in more detail.

Amino Acid Sequence↗
PubMed1985

The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction.

The in vitro splicing reactions of pre-messenger RNA (pre-mRNA) in a yeast extract were analyzed by glycerol gradient centrifugation. Labeled pre-mRNA appears in a 40S peak only if the pre-mRNA undergoes the first of the two partial splicing reactions. RNA analysis after extraction of glycerol gradient fractions shows that lariat-form intermediates, molecules that occur only in mRNA splicing, are found almost exclusively in this 40S complex. Another reaction intermediate, cut 5' exon RNA, can also be found concentrated in this complex. The complex is stable even in 400 mM KCl, although at this salt concentration, it sediments at 35S and is clearly distinguishable from 40S ribosomal subunits. This complex, termed a "spliceosome," is thought to contain components necessary for mRNA splicing; its existence can explain how separated exons on pre-mRNA are brought into contact.

Actins↗
PubMed1985

Nucleolytic processing of a tRNAArg-tRNAAsp dimeric precursor by a homologous component from Saccharomyces cerevisiae.

A subcellular extract from Saccharomyces cerevisiae has been used to transcribe cloned yeast tRNA genes in vitro and to process the primary transcripts at the 5' and 3' termini. Chromatographic fractionation of the extract has separated the transcription components from two distinct nucleolytic activities: an endonuclease that cleaves the precursors to produce mature 5' termini; and a 3'-5' exonuclease. These fractions have been used to elaborate a processing pathway for the dimeric primary transcript of the yeast tRNAArg-tRNAAsp gene pair. Under optimal conditions in vitro this gene is expressed at a rate of 200 transcripts/gene/hour, initiating at position -10 with respect to the mature 5' terminus of tRNAArg and terminating near position +160. The primary transcripts are cleaved by an endonuclease to give tRNAAsp with a mature 5' terminus, and a pre-tRNAArg monomer with a 5' leader and 3' trailer sequences. A second endonuclease cleavage of pre-tRNAArg generates the mature 5' terminus of tRNAArg. The endonuclease cleavages are not ordered. Exonuclease activity(ies) remove the spacer sequences from the 5' mature tRNAArg, and trim the 3' trailer portion from tRNAAsp. Exonucleolytic removal of the 3' trailer does not require prior endonuclease action, but removal of the spacer sequences from pre-tRNAArg is incomplete without prior removal of the 5' leader sequences.

Cell Nucleus↗
PubMed1985

Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro.

We have altered the TACTAAC sequence in the yeast CYH2m gene intron to TACTACC. This mutation changes the nucleotide at the normal position of the branch in intron RNA lariats produced during pre-mRNA splicing, and it prevents splicing in vivo. In a yeast pre-mRNA splicing system, CYH2m pre-mRNA carrying the TACTACC mutation is not specifically cut or rearranged in any way. Substitution of an A for the first G of the CYH2m intron, converting the highly conserved GTATGT 5' splice site sequence to ATATGT, also blocks intron excision in vivo and in vitro: pre-mRNA carrying this mutation was still cut normally at the mutant 5' splice site in vitro, to give authentic exon 1 and an intron-exon 2 lariat RNA with an A-A 2'-5' phosphodiester linkage at the branch point. This lariat RNA is a dead-end product. The subsequent cleavage at the 3' splice site is therefore sensitive to the sequence of the 5' end of the intron attached at the branch point.

Base Sequence↗
PubMed1984

Lariat structures are in vivo intermediates in yeast pre-mRNA splicing.

Transcripts from an ADC1-actin gene fusion containing the yeast actin intron are efficiently spliced in vivo. Four distinct forms of the excised actin intron are found in poly(A)- RNA from yeast carrying this transcription unit on a multicopy plasmid. Two of them migrate abnormally slowly in polyacrylamide gels, and one of these contains a block to reverse transcription at the conserved UACUAAC sequence (TACTAAC box). We also detect a larger RNA in the poly(A)+ fraction with abnormal gel mobility and the same reverse transcriptase block. Analysis of the major species of excised actin intron, labeled with 32P in vivo, reveals the presence of a branch at the third A in the UACUAAC sequence. This A is linked via a 3'-5' phosphodiester bond to the downstream C, and via a 2'-5' phosphodiester bond to a G, presumably from the 5' end of the intron. Thus this intron RNA is in the form of a lariat.

Actins↗
PubMed1984

Homologous in vitro transcription of linear DNA fragments containing the tRNAArg-tRNAAsp gene pair from Saccharomyces cerevisiae.

Transcription of a tRNAArg-tRNAAsp gene pair from Saccharomyces cerevisiae by an homologous yeast extract results in a dimeric percursor molecule which is processed to mature-sized tRNAArg and tRNAAsp molecules. We have transcribed linear DNA fragments cleaved within the gene sequences to show that precursor synthesis is not dependent on the internal promoter of the second gene (tRNAAsp). Furthermore, the second gene does not support independent transcription when the normal upstream initiation site is removed.

DNA, Fungal↗
PubMed1983

An RNA ligase from wheat germ which participates in transfer RNA splicing in vitro.

Transfer RNA half-molecules are intermediates in the splicing of tRNA precursors containing intervening sequences. We have utilized yeast tRNA half-molecules to identify and partially purify an ATP-dependent RNA ligase activity from extracts of wheat germ. This activity can complement a yeast tRNA endonuclease in vitro to efficiently splice 10 different yeast tRNA precursors. The products of in vitro splicing are a covalently joined tRNA and a circular intervening sequence RNA. The internucleotide bond formed at the splice junction is a 2'-phosphomonoester, 3',5'-phosphodiester structure. The 2'-phosphate originates from the 2',3'-cyclic phosphate at the 3' terminus of the 5' half-tRNA. The phosphodiester phosphate is derived from the gamma-phosphate of ATP.

Phosphorus Radioisotopes↗
PubMed1983

Enzymatic mechanism of an RNA ligase from wheat germ.

We have characterized the mechanism of action of a wheat germ RNA ligase which has been partially purified on the basis of its ability to participate in in vitro splicing of yeast tRNA precursors (Gegenheimer, P., Gabius, H-J., Peebles, C.L., and Abelson, J. (1983) J. Biol. Chem. 258, 8365-8373). The preparation catalyzes the ligation of oligoribonucleotide substrates forming a 2'-phosphomonoester, 3',5'-phosphodiester linkage. The 5' terminus of an RNA substrate can have either a 5'-hydroxyl or a 5'-phosphate. The 5'-phosphate, which for a 5'-hydroxyl substrate can be introduced by a polynucleotide kinase activity in the preparation, is incorporated into the ligated junction. The 3' terminus can have either a 2',3'-cyclic phosphate or a 2'-phosphate. 2',3'-Cyclic phosphates can be converted into 2'-phosphates by a 2',3'-cyclic phosphate, 3'-phosphodiesterase activity in the preparation. The 2'-phosphate of the ligated product is derived from the phosphate at the 3' terminus of the substrate. Ligation proceeds with the adenylylation of the 5'-phosphorylated terminus to form an intermediate with a 5',5'-phosphoanhydride bond.

Kinetics↗
PubMed1983

The yeast tRNATyr gene intron is essential for correct modification of its tRNA product.

Precise deletion of the intervening sequence of a yeast tRNATyr ochre suppressor gene (SUP6) significantly reduced its suppressor activity relative to that of the unaltered gene. This is probably the result of the absence of the pseudouridine modification, normally present at the middle anticodon position of mature suppressor tRNA, in tRNA synthesized in vivo from the deleted gene.

Anticodon↗
PubMed1983

RNA ligase in bacteria: formation of a 2',5' linkage by an E. coli extract.

Ligase activity was detected in extracts of Escherichia coli, Clostridium tartarivorum, Rhodospirillum salexigens, Chromatium gracile, and Chlorobium limicola. Ligase was measured by joining of tRNA halves produced from yeast IVS-containing tRNA precursors by a yeast endonuclease. The structure of tRNATyr halves joined by an E. coli extract was examined. The ligated junction is resistant to nuclease P1 and RNAase T2 but sensitive to venom phosphodiesterase and alkaline hydrolysis, consistent with a 2',5' linkage. The nuclease-resistant junction dinucleotide comigrates with authentic (2',5') APA marker in thin-layer chromatography. The phosphate in the newly formed phosphodiester bond is derived from the pre-tRNA substrate. The widespread existence of a bacterial ligase raises the possibility of a novel class of RNA processing reactions.

Escherichia coli↗
PubMed1983

tRNA gene transcription in yeast: effects of specified base substitutions in the intragenic promoter.

The yeast SUP53 gene encodes a leucine-inserting amber suppressor tRNA. We have introduced specific base substitutions into both 5' and 3' elements of the intragenic promoter of this tRNA gene. The influence of these sequence changes on promoter function has been investigated by transcription of the mutant genes in a homologous cell-free system. Our results do not support the idea that tertiary intragenic structure is important in tRNA gene transcription. For one of the SUP53 mutants we are able to suggest a plausible molecular basis for defective transcription: a single base substitution in the 3' element of the intragenic promoter prevents the interaction of this element with a putative transcription factor.

Base Composition↗
PubMed1983

Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease.

Splicing of transfer RNA precursors containing intervening sequences proceeds in two distinct stages: endonucleolytic cleavage, followed by ligation. We have physically separated endonuclease and ligase activities from extracts of yeast cells, and we report properties of the partially purified endonuclease preparation. The endonuclease behaves as an integral membrane protein: it is purified from a membrane fraction from which it can be solubilized with nonionic detergents, and the activity of the endonuclease in the membrane fraction is stimulated by nonionic detergents. The endonuclease cleaves precursor tRNAs at two sites to excise the intervening sequence precisely. Both the extent and the accuracy of cleavage are enhanced by the presence of spermidine; the degree of stimulation varies with the pre-tRNA substrate. The cleavage products possess 5'-hydroxyl and 2',3'-cyclic phosphodiester termini. The cyclic phosphodiester termini can be opened to 2'-phosphates by a cyclic phosphodiesterase activity in the preparation.

Base Sequence↗