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Biomedical subjects

T R Cech

Publications and source records attributed to T R Cech.

At least 199 records · Page 11Linked to original sources
PubMed1986

New reactions of the ribosomal RNA precursor of Tetrahymena and the mechanism of self-splicing.

The availability of Tetrahymena pre-rRNA of discrete size, produced by transcription of recombinant plasmids with bacteriophage SP6 RNA polymerase, has permitted a more detailed investigation of the self-splicing reaction. The predicted splicing intermediate, the product of cleavage by guanosine at the 5' splice site, was identified. This intermediate was tested in the intermolecular exon ligation reaction and found to be competent to undergo the second step of splicing. These results and others that evaluated the reactivity of the 5' and 3' splice sites independently show that splicing occurs in two separable steps. The 3' splice site was found to be susceptible to site-specific hydrolysis leaving a hydroxyl terminus. This is interpreted as an indication that the 3' splice site is activated for nucleophilic attack in general and for exon ligation in particular. Preliminary evidence for specific hydrolysis at the 5' splice site was also obtained. All of the newly characterized intervening sequence RNA-mediated reactions as well as those found previously are divided into three categories: transesterification by guanosine at sites following two or three pyrimidine nucleotides (and, as a minor reaction, at sites following other guanosine residues); transesterification by oligopyrimidines or by the 5' exon (which terminates with C-U-C-U-C-UOH) at the site following the 3'-terminal guanosine residue of the intervening sequence; and specific hydrolysis at the splice sites. One of the products of the reactions at the 3' splice site is a molecule that contains the 5' exon still attached to the intervening sequence. It has a 3'-terminal GOH and undergoes cyclization both at the normal cyclization site within the intervening sequence and at the 5' splice site. The finding that the splice site can act as a cyclization site, combined with the earlier observation that the normal cyclization site is subject to attack by guanosine mononucleotide, leads us to propose that all these reactions may be occurring in the same active site. Translocation (a conformational change) would then bring different oligopyrimidine sequences into the active site for attack by guanosine. On the basis of the experimental results, a model for the local structure at the active site is described. A key feature of the model is the interaction between the U at the end of the oligopyrimidine sequence, a G residue within the internal guide sequence in the intervening sequence, and another G residue that can be either the attacking group for transesterification or the 3'-terminal G of the intervening sequence.

Animals↗
PubMed1986

Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor.

Oligonucleotide-directed mutagenesis has been used to alter highly conserved sequences within the intervening sequence (IVS) of the Tetrahymena large ribosomal RNA precursor. Mutations within either sequence element 9L or element 2 eliminate splicing activity under standard in vitro splicing conditions. A double mutant with compensatory base changes in elements 9L and 2 has accurate splicing activity restored. Thus, the targeted nucleotides of elements 9L and 2 base-pair with one another in the IVS RNA, and pairing is important for self-splicing. Mutant splicing activities are restored by increased magnesium ion concentrations, supporting the conclusion that the role of the targeted bases in splicing is primarily structural. Based on the temperature dependence, we propose that a conformational switch involving pairing and unpairing of elements 9L and 2 is required for splicing.

Animals↗
PubMed1986

The intervening sequence RNA of Tetrahymena is an enzyme.

A shortened form of the self-splicing ribosomal RNA (rRNA) intervening sequence of Tetrahymena thermophila acts as an enzyme in vitro. The enzyme catalyzes the cleavage and rejoining of oligonucleotide substrates in a sequence-dependent manner with Km = 42 microM and kcat = 2 min-1. The reaction mechanism resembles that of rRNA precursor self-splicing. With pentacytidylic acid as the substrate, successive cleavage and rejoining reactions lead to the synthesis of polycytidylic acid. Thus, the RNA molecule can act as an RNA polymerase, differing from the protein enzyme in that it uses an internal rather than an external template. At pH 9, the same RNA enzyme has activity as a sequence-specific ribonuclease.

Animals↗
PubMed1986

RNA as an enzyme.

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Catalysis↗
PubMed1986

A model for the RNA-catalyzed replication of RNA.

A shortened form of the self-splicing ribosomal RNA intervening sequence of Tetrahymena thermophila has enzymatic activity as a poly(cytidylic acid) polymerase [Zaug, A.J. & Cech, T.R. (1986) Science 231, 470-475]. Based on the known properties of this enzyme, a detailed model is developed for the template-dependent synthesis of RNA by an RNA polymerase itself made of RNA. The monomer units for RNA synthesis are tetra- and pentanucleotides of random base sequence. Polymerization occurs in a 5'-to-3' direction, and elongation rates are expected to approach two residues per minute. If the RNA enzyme could use another copy of itself as a template, RNA self-replication could be achieved. Thus, it seems possible that RNA catalysts might have played a part in prebiotic nucleic acid replication, prior to the availability of useful proteins.

Catalysis↗
PubMed1985

Sites of circularization of the Tetrahymena rRNA IVS are determined by sequence and influenced by position and secondary structure.

The sequence of the cloned Tetrahymena ribosomal RNA intervening sequence (IVS) was altered at the site to which circularization normally occurs. The alterations caused circularization to shift to other sites, usually a nearby position which followed three pyrimidines. While a tripyrimidine sequence was the major determinant of a circularization site, both location of a sequence and local secondary structure may influence the use of that sequence. For some constructs circularization appeared to occur at the position following the 5' G, the nucleotide added to the IVS during its excision. Portions of the internal guide sequence (IGS), proposed to interact with the 3'exon were deleted without preventing exon ligation. Thus if the IGS-3'exon interaction exists, it is not essential for splicing in vitro.

Animals↗
PubMed1985

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by intercalating dyes.

The intervening sequence (IVS) excised from the pre-rRNA of Tetrahymena undergoes a self-catalyzed cleavage-ligation reaction to form a covalently closed circular RNA. This cyclization reaction is kinetically inhibited by ethidium bromide (50% inhibition at 22 +/- 14 microM, greater than 99% inhibition at 53 +/- 16 microM for a 20 minute reaction). The dye does not alter the sites of the cyclization reaction, but it does increase the relative amount of reaction at a minor site 19 nucleotides from the 5' end of the IVS. The reversibility of the inhibition and the relative inhibitory strength of acridine orange, ethidium and proflavine suggest that inhibition is due to intercalation of the dye in functionally important secondary or tertiary structures of the IVS. The concentration of dye required to inhibit cyclization is much higher than expected from the known binding constants of such dyes to tRNA. At high Mg2+ to Na+ ratios, conditions which should stabilize RNA structure, a subpopulation of the IVS RNA molecules is resistant to ethidium inhibition, even at 200 microM ethidium. These data are interpreted as reflecting two conformational isomers of the IVS that differ in their reactivity and in their sensitivity to dye binding.

Acridine Orange↗
PubMed1985

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by methidiumpropyl.EDTA and localization of the major dye binding sites.

The intervening sequence (IVS) excised from the rRNA precursor of Tetrahymena thermophila is converted to a covalently closed circular RNA in the absence of proteins in vitro. This self-catalyzed cyclization reaction is inhibited by the intercalating dye methidiumpropyl.EDTA (MPE; R.P. Hertzberg and P.B. Dervan (1982) J. Am. Chem. Soc. 104, 313-315). The MPE binding sites have been localized by mapping the sites of MPE.Fe(II) cleavage of the IVS RNA. There are three major binding sites within the 414 nucleotide IVS RNA. Two of these sites coincide with the A.B and 9L.2 pairings. These are structural elements that are conserved in all group I introns and are implicated as being functionally important for splicing. We propose that interaction of MPE with these sites is responsible for dye inhibition of cyclization. The reactions of MPE.Fe(II) with an RNA of known structure, tRNAPhe, and with the IVS RNA were studied as a function of temperature, ionic strength and ethidium concentration. Based on the comparison of the reaction with these two RNAs, we conclude that the dye is a very useful probe for structural regions of large RNAs, while it provides more limited structural information about the small, compact tRNA molecule.

Animals↗
PubMed1985

Reactions of the intervening sequence of the Tetrahymena ribosomal ribonucleic acid precursor: pH dependence of cyclization and site-specific hydrolysis.

During self-splicing of the Tetrahymena rRNA precursor, the intervening sequence (IVS) is excised as a unique linear molecule and subsequently cyclized. Cyclization involves formation of a phosphodiester bond between the 3' end and nucleotide 16 of the linear RNA, with release of an oligonucleotide containing the first 15 nucleotides. We find that the rate of cyclization is independent of pH in the range 4.7-9.0. A minor site of cyclization at nucleotide 20 is characterized. Cyclization to this site becomes more prominent at higher pHs, although under all conditions examined it is minor compared to cyclization at nucleotide 16. The circular IVS RNAs are unstable, undergoing hydrolysis at the phosphodiester bond that was formed during cyclization. We find that the rate of site-specific hydrolysis is first order with respect to hydroxide ion concentration, with a rate constant 10(3)-10(4)-fold greater than that of hydrolysis of strained cyclic phosphate esters. On the basis of these results, we propose that circular IVS RNA hydrolysis involves direct attack of OH- on the phosphate at the ligation junction, that particular phosphate being made particularly reactive by the folding of the RNA molecule. Cyclization, on the other hand, appears to occur by direct attack of the 3'-terminal hydroxyl group of the linear IVS RNA without prior deprotonation.

Animals↗
PubMed1985

Oligomerization of intervening sequence RNA molecules in the absence of proteins.

The intervening sequence RNA excised from the ribosomal RNA precursor of Tetrahymena forms linear and circular oligomers when exposed to a heating-cooling treatment in vitro. The reactions require no protein or external energy source. Oligomerization is different from other self-catalyzed reactions of the intervening sequence RNA in that it involves intermolecular rather than intramolecular recombination, producing RNA molecules that are substantially larger than the original. The observation that RNA molecules can catalyze their own oligomerization has possible implications for the evolution of chromosomes and for the replicative cycle of plant viroids and virus-associated RNA's.

Animals↗
PubMed1985

Coupling of Tetrahymena ribosomal RNA splicing to beta-galactosidase expression in Escherichia coli.

Splicing of the Tetrahymena ribosomal RNA precursor is mediated by the folded structure of the RNA molecule and therefore occurs in the absence of any protein in vitro. The Tetrahymena intervening sequence (IVS) has been inserted into the gene for the alpha-donor fragment of beta-galactosidase in a recombinant plasmid. Production of functional beta-galactosidase is dependent on RNA splicing in vivo in Escherichia coli. Thus RNA self-splicing can occur at a rate sufficient to support gene expression in a prokaryote, despite the likely presence of ribosomes on the nascent RNA. The beta-galactosidase messenger RNA splicing system provides a useful method for screening for splicing-defective mutations, several of which have been characterized.

Base Sequence↗
PubMed1985

Sequence requirements for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA.

The sequence requirements for splicing of the Tetrahymena pre-rRNA have been examined by altering the rRNA gene to produce versions that contain insertions and deletions within the intervening sequence (IVS). The altered genes were transcribed and the RNA tested for self-splicing in vitro. A number of insertions (8-54 nucleotides) at three locations had no effect on self-splicing activity. Two of these insertions, located at a site 5 nucleotides preceding the 3'-end of the IVS, did not alter the choice of the 3' splice site. Thus the 3' splice site is not chosen by its distance from a fixed point within the IVS. Analysis of deletions constructed at two sites revealed two structures, a hairpin loop and a stem-loop, that are entirely dispensable for IVS excision in vitro. Three other regions were found to be necessary. The regions that are important for self-splicing are not restricted to the conserved sequence elements that define this class of intervening sequences. The requirement for structures within the IVS for pre-rRNA splicing is in sharp contrast to the very limited role of IVS structure in nuclear pre-mRNA splicing.

Base Sequence↗
PubMed1985

Reversibility of cyclization of the Tetrahymena rRNA intervening sequence: implication for the mechanism of splice site choice.

The Tetrahymena rRNA intervening sequence (IVS) excises itself from the pre-rRNA and then mediates its own cyclization. We now find that certain di- and trinucleotides with free 3' hydroxyl groups reopen the circular IVS at the cyclization junction, producing a linear molecule with the oligonucleotide covalently attached to its 5' end. This linear molecule recyclizes with release of the added oligonucleotide. Thus the IVS RNA, like an enzyme, lowers the activation energy for both forward and reverse cleavage-ligation reactions. Certain combinations of pyrimidines are required for circle reopening. The most reactive oligonucleotide is UCU. This sequence resembles those preceding the major and minor cyclization sites in the linear IVS RNA (UUU and CCU) and the 5' splice site in the pre-rRNA (UCU). We propose that an oligopyrimidine binding site within the IVS binds the sequences upstream of each of these target sites for cleavage-ligation.

Animals↗
PubMed1985

Intermolecular exon ligation of the rRNA precursor of Tetrahymena: oligonucleotides can function as 5' exons.

The dinucleotide CpUOH, when incubated with self-splicing Tetrahymena pre-rRNA in the absence of GTP, functions as a 5' exon. It cleaves the precursor exactly at the 3' splice site and becomes covalently ligated to the 3' exon. Other oligonucleotides with sequences that resemble CUCUCU, the sequence at the 3' end of the 5' exon, can add to the 3' exon in this reaction. Such splicing in trans is most readily explained by a site within the intervening sequence that binds the last few nucleotides of the 5' exon. This binding site functions in splice site recognition and is also part of the active site of the ribozyme. The mechanism by which 5' splice sites are selected in Tetrahymena rRNA and group I mitochondrial RNA splicing is like that used in nuclear mRNA splicing, in that it involves specific pairing of bases adjacent to the splice site with a complementary RNA sequence.

Animals↗
PubMed1985

Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase.

The structure of the intervening sequence (IVS) of the Tetrahymena rRNA precursor mediates cleavage-ligation reactions that result in pre-rRNA splicing and IVS cyclization. We have developed a method for RNA structure analysis and applied it to the circular form of the IVS RNA. The native RNA was treated with dimethyl sulfate or diethyl pyrocarbonate to modify bases not involved in secondary or tertiary interactions. The RNA was then used as a template for reverse transcription. Elongation of synthetic oligodeoxynucleotide primers was found to stop (or pause) one nucleotide prior to 1-methyladenosine, 3-methylcytidine, and 7-ethoxycarbonyladenosine residues. The detection of 1-methyladenosine is particularly useful for locating single-stranded regions. After chemical cleavage of the RNA, 7-methylguanosine also could be detected. In general, the sites of modification were consistent with a previous model of the secondary structure of the linear form of the IVS RNA, a model based on enzymatic cleavage data, free energy calculations, and phylogenetic comparison. Thus, IVS RNA autocyclization does not involve major rearrangements of the secondary structure, although there is evidence for a conformational change in one region of the molecule. The methods described here should be of general use for obtaining information about structure far from the ends of RNA molecules.

Animals↗