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T R Cech

Publications and source records attributed to T R Cech.

At least 217 records · Page 12Linked to original sources
PubMed1984

A labile phosphodiester bond at the ligation junction in a circular intervening sequence RNA.

The excised intervening sequence of the Tetrahymena ribosomal RNA precursor mediates its own covalent cyclization in the absence of any protein. The circular molecule undergoes slow reopening at a single phosphodiester bond, the one that was formed during cyclization. The resulting linear molecule has 5'-phosphate and 3'-hydroxyl termini; these are unusual products for RNA hydrolysis but are typical of the other reactions mediated by this molecule. The reopened circle retains cleavage-ligation activity, as evidenced by its ability to undergo another round of cyclization and reopening. The finding that an RNA molecule can be folded so that a specific phosphate can be strained or activated helps to explain how the activation energy is lowered for RNA self-splicing. The proposed mechanisms may be relevant to several other RNA cleavage reactions that are RNA-mediated.

Animals↗
PubMed1984

DNA-protein interactions in the Drosophila virilis mitochondrial chromosome.

The location of proteins on the mitochondrial DNA (mtDNA) of Drosophila virilis was investigated by Me3 psoralen photoreaction of mitochondria isolated from embryos. After photoreaction the mtDNA was purified and the pattern of DNA cross-linking was determined by electron microscopy of the DNA under totally denaturing conditions. The transcribed regions of the mtDNA molecule contained some uncross-linked regions, but such regions were infrequent and randomly distributed. In contrast, the A + T-rich region around the origin of replication of the mtDNA was usually protected from psoralen cross-linking. The data were best fit by two protected sites, each approximately 400 base pairs, compared to the four 400 base pair sites observed in the equivalent region of D. melanogaster mtDNA [Potter et al. (1980) Proc. Nat. Acad. Sci. USA 77, 4118-4122]. Thus this region of the mtDNA appears to be involved in a DNA-protein structure that is highly conserved even though the DNA sequence has diverged rapidly relative to protein-coding sequences.

Animals↗
PubMed1984

Chromatin structure at the replication origins and transcription-initiation regions of the ribosomal RNA genes of Tetrahymena.

The chromatin structure of regulatory regions of the extrachromosomal rRNA genes of Tetrahymena thermophila was probed by nuclease treatment of isolated nuclei. The chromatin near the origins of replication contains hypersensitive sites for micrococcal nuclease, DNAase I, and DNAase II. These sites persist in starved cells, consistent with the origins' being maintained in an altered chromatin structure independent of DNA replication. The region between the two origins of replication is organized into a phased array of seven nucleosomes, the fourth of which is centered at the axis of symmetry of the palindromic rDNA. The entire transcribed region and 150 bp upstream from the initiation site are generally accessible to nucleases; any histone proteins associated with these regions are clearly not in a highly organized nucleosomal array as seen in the central region. Comparison of the chromatin structures of the central spacer of T. thermophila and T. pyriformis rDNA reveals that deletion or insertion of DNA has occurred in increments of 200 bp. This is taken to imply that there are constraints on the evolution of spacer DNA sequences at the level of the nucleosome.

Animals↗
PubMed1984

Chromatin structure of the molecular ends of Oxytricha macronuclear DNA: phased nucleosomes and a telomeric complex.

Oxytricha macronuclear DNA exists as approximately 24 X 10(6) gene-sized molecules terminating with a C4A4 repeat. DNA-protein interactions at the ends of bulk macronuclear molecules were probed with micrococcal nuclease and methidiumpropyl-EDTA X Fe(II) (MPE X Fe[II]). The ends were indirectly labeled by hybridizing with (C4A4)2. Alternatively, a novel method using MPE X FE(II) as a probe and directly labeling the 3' ends with terminal transferase was implemented. A terminal complex involving approximately 100 bp with nucleosomes phased inward from the complex was found to be characteristic of most or all of the ends. Analysis of two specific genes confirmed the pattern and showed that the special structure was on both ends of each molecule. We conclude that a DNA-protein complex involving 100 bp and terminating with the C4A4 repeat can be sufficient to provide the fundamental functions of telomeres, allowing linear DNA replication and conferring stability of linear DNA.

Animals↗
PubMed1983

Fate of an intervening sequence ribonucleic acid: excision and cyclization of the Tetrahymena ribosomal ribonucleic acid intervening sequence in vivo.

In previous studies of RNA splicing in vitro, we have shown that the intervening sequence (IVS) of the Tetrahymena rRNA precursor is excised as a unique linear RNA molecule and subsequently cyclized. In the present work, we have investigated the occurrence and stability of these RNA species in vivo. RNA was separated by gel electrophoresis, transferred to diazotized paper, and hybridized with 32P-labeled DNA probes. RNA molecules containing the IVS were found to reside within the nucleus and not in the cytoplasm. The species found in nucleus include both the linear and circular forms of the excised IVS RNA, as well as the unspliced precursor. On the basis of quantitation of the hybridization, the half-lives of the IVS-containing pre-rRNA and the excised IVS RNA in rapidly growing cells were estimated as 2 and 6 s, respectively. We conclude that splicing is not a rate-limiting step in rRNA maturation and that the IVS RNA is quickly degraded after its excision. When the deproteinized nuclear RNA was incubated at 37 degrees C in a Mg2+-containing solution, a substantial portion of the linear IVS RNA was converted to the circular form. Autocyclization, previously characterized with IVS RNA produced by splicing in vitro, is therefore also a property of IVS RNA produced in vivo.

Animals↗
PubMed1983

Transcribed and non-transcribed regions of Tetrahymena ribosomal gene chromatin have different accessibilities to micrococcal nuclease.

DNA renaturation kinetics was used to examine the relative accessibility of various regions of the Tetrahymena ribosomal RNA gene (rDNA) chromatin to micrococcal nuclease. In nuclei from cells active in rRNA transcription, the transcribed region of the rDNA chromatin was as much as 5-fold more accessible than the average of the total chromatin. As few as 20% inactive genes in the population could have accounted for all of the hybridization, so the transcribed region of the active units may be totally unprotected from nuclease degradation. The terminal non-transcribed spacer downstream from the transcription unit was also preferentially digested, but to a smaller degree. The central non-transcribed spacer was degraded to the same extent as total chromatin after a high degree of nuclease digestion. In nuclei from starved cells, which have 96% reduced rRNA transcription, the transcribed and terminal spacer regions of the rDNA were again more accessible than the total chromatin from the same nuclei, but the difference did not exceed 2-fold. We conclude that transcriptional activation is accompanied by major changes in the structure of the ribosomal gene chromatin, and that the extent and/or type of structural alteration differs in each functionally defined region of the rDNA.

Animals↗
PubMed1983

Different nucleosome spacing in transcribed and non-transcribed regions of the ribosomal RNA gene in Tetrahymena thermophila.

The chromatin structure of the palindromic macronuclear ribosomal RNA genes of Tetrahymena thermophila was probed with micrococcal nuclease. Independent of the state of transcriptional activity, the transcribed region had a shorter nucleosome repeat (184 +/- 3 base pairs) than the non-transcribed central spacer or bulk chromatin (both 200 base pairs). The transcribed region displayed an increased sensitivity to micrococcal nuclease in rapidly growing cells, which suggested an altered chromatin structure during transcription. At early stages of nuclease digestion, the central spacer appeared to be in a highly structured nucleosomal array. Based on the differences in nucleosome repeat distance and sensitivity to nuclease, we conclude that quite different chromatin structures are maintained in two adjacent regions of the Tetrahymena ribosomal RNA gene. The DNA of the non-transcribed terminal spacer was found to contain sequences which are highly susceptible to micrococcal nuclease, precluding any conclusions about nucleosome structure in this region.

Animals↗
PubMed1983

Secondary structure of the Tetrahymena ribosomal RNA intervening sequence: structural homology with fungal mitochondrial intervening sequences.

Splicing of the ribosomal RNA precursor of Tetrahymena is an autocatalytic reaction, requiring no enzyme or other protein in vitro. The structure of the intervening sequence (IVS) appears to direct the cleavage/ligation reactions involved in pre-rRNA splicing and IVS cyclization. We have probed this structure by treating the linear excised IVS RNA under nondenaturing conditions with various single- and double-strand-specific nucleases and then mapping the cleavage sites by using sequencing gel electrophoresis. A computer program was then used to predict the lowest-free-energy secondary structure consistent with the nuclease cleavage data. The resulting structure is appealing in that the ends of the IVS are in proximity; thus, the IVS can help align the adjacent coding regions (exons) for ligation, and IVS cyclization can occur. The Tetrahymena IVS has several sequences in common with those of fungal mitochondrial mRNA and rRNA IVSs, sequences that by genetic analysis are known to be important cis-acting elements for splicing of the mitochondrial RNAs. In the predicted structure of the Tetrahymena IVS, these sequences interact in a pairwise manner similar to that postulated for the mitochondrial IVSs. These findings suggest a common origin of some nuclear and mitochondrial introns and common elements in the mechanism of their splicing.

Base Sequence↗
PubMed1982

The intervening sequence excised from the ribosomal RNA precursor of Tetrahymena contains a 5-terminal guanosine residue not encoded by the DNA.

The ribosomal RNA precursor of Tetrahymena thermophila contains a 0.4 kilobase intervening sequence that is excised as a linear RNA molecule ("IVS RNA") and subsequently cyclized. In vitro transcription in isolated nuclei was used to accumulate the IVS RNA labeled at its 5' end was subjected to sequencing gel analysis and terminal nucleotide analysis. In addition, uniformly labeled IVS RNA was cleaved with RNAase T1, and the resulting oligonucleotides were studied by two-dimensional fingerprinting. The IVS RNA was found to be a unique molecule with no discernible terminal heterogeneity. The 5'-terminal nucleotide is a guanosine that is not present at the corresponding point in the DNA sequence, determined by N. Kan and J. Gall (see adjoining paper). This nucleotide is added to the IVS during splicing [Cech, Zaug, and Grabowski (1981) Cell 27, 487-496]. Based on the sequences near the ends of the RNA, the remainder of the RNA sequence is colinear with that of the DNA. The IVS RNA has 5'-monophosphate and 3'-hydroxyl termini. Comparison of these results to those obtained previously for yeast tRNA intervening sequences leads us to conclude that the splicing mechanisms are fundamentally different for these two classes of transcripts.

Animals↗
PubMed1982

Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena.

In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region. A restriction fragment of the rDNA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UV5 promoter in a recombinant plasmid. Transcription of this template by purified Escherichia coli RNA polymerase in vitro produced a shortened version of the pre-rRNA, which was then deproteinized. When incubated with monovalent and divalent cations and a guanosine factor, this RNA underwent splicing. The reactions that were characterized included the precise excision of the IVS, attachment of guanosine to the 5' end of the IVS, covalent cyclization of the IVS and ligation of the exons. We conclude that splicing activity is intrinsic to the structure of the RNA, and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions. We propose that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds. The finding of autocatalytic rearrangements of RNA molecules has implications for the mechanism and the evolution of other reactions that involve RNA.

Animals↗
PubMed1981

Replication of the extrachromosomal ribosomal RNA genes of Tetrahymena thermophilia.

Cultures of Tetrahymena thermophila were deprived of nutrients and later refed with enriched medium to obtain partial synchrony of DNA replication. Preferential replication of the extrachromosomal, macronuclear ribosomal RNA genes (rDNA) was found to occur at 40-80 min after refeeding. The rDNA accounted for one half of the label incorporated into cellular DNA during this period. Electron microscopy of the purified rDNA showed 1% replicative intermediates. Their structure was that expected for bidirectional replication of the linear rDNA from an origin or origins located in the central nontranscribed region of the palindromic molecule. Similar forms had previously been observed for the rDNA of a related species, Tetrahymena pyriformis. The electron microscopic data was consistent with an origin of replication located approximatley 600 base pairs from the center of the rDNA of T. thermophila, in contrast to a more central location in the rDNA of T. pyriformis. One implication of an off-center origin of replication is that there are two such sequences per palindromic molecule.

Animals↗
PubMed1981

Alkaline gel electrophoresis of deoxyribonucleic acid photoreacted with trimethylpsoralen: rapid and sensitive detection of interstrand cross-links.

Restriction fragments of phage lambda and phi X174 deoxyribonucleic acid (DNA) were photoreacted with 4,5',8-trimethylpsoralen to various extents, and the amount of covalent cross-linking was determined by electron microscopy of the DNA under totally denaturing conditions. The DNA was then analyzed by electrophoresis in alkaline agarose gels. A single cross-link in a DNA molecule produced a large decrease in its electrophoretic mobility. With DNA fragments 0.3--4 kilobase pairs in size, the apparent Mr (molecular weight) of the cross-linked DNA was 2.0 +/- 0.1 times and Mr of the unreacted, single-stranded DNA. A single cross-link in a larger DNA molecule resulted in an even greater increase in apparent Mr. Further cross-linking produced a decrease in the apparent Mr of the DNA, reaching a plateau at a value of 1.4 +/- 0.1 times the Mr of the unreacted, single-stranded DNA over a large range of fragment sizes (0.6--10 kilobase pairs). The apparent Mr of the cross-linked DNA was weakly dependent on the percentage of agarose in the gel. Although highly sensitive to interstrand cross-links the electrophoretic mobilities appeared to be unaffected by low levels of monoadducts (trimethylpsoralen covalently bound to one strand of the DNA). The DNA bandwidths increased by as much as 4-fold at low extents of cross-linking, presumably due to heterogeneity in the locations of the cross-links in the DNA molecules. The bands became sharp again at high levels of reaction. These observations from the basis of a new assay for interstrand DNA cross-links that is both more sensitive and more convenient than previous methods.

Bacteriophage lambda↗
PubMed1981

The intervening sequence of the ribosomal RNA precursor is converted to a circular RNA in isolated nuclei of Tetrahymena.

The Tetrahymena thermophila ribosomal RNA gene contains an intervening sequence (IVS), which is transcribed as part of the precursor RNA and subsequently removed by splicing. We have found previously that the IVS is excised as a 0.4 kb RNA in isolated nuclei. We now report the finding of a novel RNA molecule, which is an electrophoretic variant (EV) of this 0.4 kb IVS RNA. The EV was identified as a form of the IVS RNA by Southern hybridization, RNA fingerprinting and R-loop mapping. A pulse-chase experiment established that in vitro the excised IVS RNA is converted to the EV by a post-splicing event. This conversion is enhanced at 39 degrees C compared to 30 degrees C and is irreversible under our experimental conditions. The EV of the IVS is a circular RNA. This structure was first suggested by its anomalous electrophoretic mobility on denaturing compared to nondenaturing gels. When the EV was prepared for electron microscopy under totally denaturing conditions, 0.4 kb circular molecules were observed. Furthermore, we have converted the circular form to a linear form by limited T1 RNAase digestion. The circular RNA survived treatment with DNAase, protease, glyoxal and various denaturants, which suggests that it is a covalently closed RNA circle.

Animals↗
PubMed1981

In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence.

In previous studies of transcription and splicing of the ribosomal RNA precursor in isolated Tetrahymena nuclei, we found that the intervening sequence (IVS) was excised as a unique linear RNA molecule and was subsequently cyclized. In the present work, transcription at low monovalent cation concentration is found to inhibit splicing and to lead to the accumulation of a splicing intermediate. This intermediate contains splicing activity that either is tightly bound to the RNA or is part of the RNA molecule itself. The intermediate is able to complete the excision of the IVS when it is incubated with a monovalent cation (75 mM (NH4)2SO4), a divalent cation (5-10 mM MgCl2) and a guanosine compound (1 microM GTP, GDP, GMP or guanosine). ATP, UTP, CTP and guanosine compounds without 2' and 3' hydroxyl groups are inactive in causing excision of the IVS. Accurate excision of the IVS, cyclization of the IVS and (apparently) ligation of the 26S rRNA sequences bordering the IVS all take place under these conditions, suggesting that a single activity is responsible for all three reactions. During excision of the IVS, the 3' hydroxyl of the guanosine moiety becomes linked to the 5' end of the IVS RNA via a normal phosphodiester bond. When GTP is used to drive the reaction, it is added intact without hydrolysis. Based on these results, we propose that Tetrahymena pre-rRNA splicing occurs by a phosphoester transferase mechanism. According to this model, the guanosine cofactor provides the free 3' hydroxyl necessary to initiate a series of three transfers that results in splicing of the pre-rRNA and cyclization of the excised IVS.

Animals↗
PubMed1980

In vitro splicing of the ribosomal RNA precursor in nuclei of Tetrahymena.

The macronuclear rRNA genes of Tetrahymena thermophila contain a 0.4 kb intervening sequence. In this paper we present evidence that the excision of the intervening sequence from pre-rRNA occurs in vitro in isolated T. thermophila nuclei. The transcription-processing system includes alpha-amanitin to inhibit non-rRNA synthesis and aurintricarboxylic acid to inhibit endogenous nucleases. A discrete 0.4 kb RNA comprises up to 6% of the RNA synthesized in this system. Southern hybridization with restriction fragments of the rDNA (rRNA genes) shows that the 0.4 kb RNA contains the intervening sequence. The size of the 0.4 kb RNA, 410-425 nucleotides, is the size predicted if the entire intervening sequence were excised as a single linear molecule. The 0.4 kb RNA accumulates post-transcriptionally and appears to be stable in vitro. T. pigmentosa strain 6UM, whose rDNA has an intervening sequence of the same size and location as that of T. thermophila, also produces a 0.4 kb RNA in vitro; T. pigmentosa strain 8ALP, with not rDNA intervening sequence, produces no such RNA. The measurement of the accumulation of the excised intervening sequence is a convenient assay for the pre-rRNA splicing activity and a means for characterizing some of the details of the reaction. The resistance of the splicing acticity to concentrations of aurintricarboxylic acid that inhibit other endogenous nucleases should be useful in assaying the splicing enzyme during purification.

Animals↗
PubMed1980

DNA-protein interactions in the Drosophila melanogaster mitochondrial genome as deduced from trimethylpsoralen crosslinking patterns.

The location of proteins on the mitochondrial DNA (mtDNA) of Drosophila melanogaster was investigated by trimethylpsoralen photoreaction of embryos disrupted by gentle homogenization. After photoreaction, the mtDNA was isolated and the pattern of DNA crosslinking was determined by electron microscopy of the DNA under totally denaturing conditions. In contrast to nuclear DNA, which showed periodic crosslinks indicative of a nucleosome structure, most of each mtDNA molecule exhibited uniformly heavy crosslinking. A 10% region of the mtDNA was, however, protected from psoralen crosslinking in a distinctive manner: five uncrosslinked segments were closely clustered in the mtDNA. Four were 394 +/- 13 (SD) base pairs in size, while the fifth measured about 200 base pairs. These protected segments mapped within the A+T-rich region of the mtDNA, extending from the end of the A+T-rich region near the Bg1 II cleavage site to the center of the A+T-rich region. Protection of this part of the mtDNA from crosslinking was interpreted to be the result of association with proteins in the mitochondrion because mtDNA that was deproteinized before the photoreaction was uniformly crosslinked over its entire length. The origin of replication of the mtDNA is also located at the center of the A+T-rich region, which suggests that the protection from the psoralen photoreaction may be due to proteins involved in membrane attachment or replication.

Animals↗