Self-cleavage Reaction Research Articles

Conformation-dependent cleavage of staphylococcal nuclease with a disulfide-linked iron chelate.

We report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide. By using this easily prepared cysteine-specific hydrophilic reagent, an ethylenediaminetriacetic acid-Fe3+ complex (EDTA-Fe) was covalently attached to a single genetically engineered cysteine residue in staphylococcal nuclease. Upon addition of the iron reductant ascorbate, the nuclease-EDTA-Fe conjugate underwent a protein self-cleavage reaction mediated by reactive oxygen species. Sequence analysis of the products indicated that cleavage occurs close in tertiary structure to the EDTA-Fe attachment site. In the presence of denaturants, the cleavage pattern changes and the reaction is limited to residues proximal in sequence to the cysteine attachment site. These results indicate that intramolecular protein cleavage reactions mediated by EDTA-Fe can be used to evaluate changes in protein conformation. The reagent described should be a useful tool in the structural mapping of nonnative protein states populated at equilibrium, such as the molten globule, that are frequently refractory to conventional structure analysis.

The strands of both polarities of a small circular RNA from carnation self-cleave in vitro through alternative double- and single-hammerhead structures.

The sequence of a circular RNA from carnation has been determined and found to consist of 275 nucleotide residues adopting a branched secondary structure of minimum free energy. Both plus and minus strands of this RNA can form the hammerhead structures proposed to mediate the in vitro self-cleavage of a number of small infectious plant RNAs and the transcript of satellite 2 DNA from the newt. Minus full- and partial-length transcripts of the carnation circular RNA including the hammerhead structure showed self-cleavage during transcription and after purification, indicating the involvement of a single-hammerhead structure in the self-cleavage reaction. In the case of the plus transcripts only a dimeric RNA, but not a monomeric one, self-cleaved efficiently during transcription and after purification, strongly supporting the implication in this process of a double-hammerhead structure theoretically more stable than the corresponding single cleavage domain. However, a plus monomeric transcript self-cleaved after purification at a slow rate in a concentration-independent reaction which most probably occurs through an intramolecular mechanism. Comparative sequence analysis has revealed that the circular RNA from carnation shares similarities with some representative members of the viroid and viroid-like satellites RNAs from plants, suggesting that it is a new member of either these two groups of small pathogenic RNAs.

Regulation of polyadenylation of hepatitis delta virus antigenomic RNA

Hepatitis delta virus (HDV) is a subviral agent with a small RNA genome that is replicated in the nucleus of an infected cell. During genome replication, there is the synthesis of a complementary RNA, known as the antigenome, and also of a smaller complementary species that is polyadenylated and acts in the cytoplasm as the mRNA for the only known HDV protein, the delta antigen. We have carried out an examination of the cis- and trans-acting elements that regulate the polyadenylation process involved in the synthesis of this mRNA for the delta antigen. Our experimental approach has been to study the processing of nascent antigenomic RNA as it occurs in transfected cells via DNA-directed RNA synthesis, in the absence of genome replication. Three conclusions have been made. (i) The polyadenylation process occurs independent of the functionality of a unique self-cleavage domain located just 3' of the polyadenylation site. (ii) RNA transcripts that proceed beyond the polyadenylation site can be stabilized by the self-cleavage reaction. Thus, a single transcription initiation event can lead not only to the mRNA species but also to at least one more stable RNA species. (iii) If the nascent RNA species can fold on itself, into the so-called rodlike structure, then the presence of the delta antigen leads to a major suppression of polyadenylation. These results are incorporated into a more detailed model of the replication of the HDV genome.

Alternative hammerhead structures in the self-cleavage of avocado sunblotch viroid RNAs

The plus and minus RNAs of the 247 nt avocado sunblotch viroid (ASBV) undergo site specific RNA self-cleavage reactions in vitro. As with several other self-cleaving RNAs, we proposed hammerhead secondary structures for the sequence around the site of self-cleavage of both RNAs. We have shown previously that, during transcription of a dimeric plus ASBV RNA, a double-hammerhead structure formed and was necessary for self-cleavage. Here, we show that the purified full-length dimeric plus RNA, when incubated under our standard self-cleavage conditions, also self-cleaved by a double-hammerhead structure. In contrast, a dimeric minus ASBV RNA self-cleaved by a double-hammerhead structure during transcription, but by a single-hammerhead structure after purification. This illustrates the importance of the pathway of folding of the RNA in determining which active self-cleaving structure is formed.

Characterization of deoxy- and ribo-containing oligonucleotide substrates in the hammerhead self-cleavage reaction

Deoxynucleotides were introduced into a substrate fragment of the hammerhead RNA self-cleaving domain. A substrate lacking the 2′ hydroxyl adjacent to the cleavage site showed no detectable cleavage under a variety of reaction conditions. Competition experiments indicate that this fragment binds to the ribozyme with an affinity similar to the all RNA fragment, suggesting that the attacking 2′ hydroxyl does not substantially contribute to the binding of substrate to ribozyme. Similar competition experiments with the all DNA substrate indicate a much lower affinity for the ribozyme perhaps due to the lack of other 2′ hydroxyls. A substrate containing all deoxy residues except for a ribonucleotide at the cleavage site was also shown to be active.

Sequence requirements of the hammerhead RNA self-cleavage reaction

A previously well-characterized hammerhead catalytic RNA consisting of a 24-nucleotide substrate and a 19-nucleotide ribozyme was used to perform an extensive mutagenesis study. The cleavage rates of 21 different substrate mutations and 24 different ribozyme mutations were determined. Only one of the three phylogenetically conserved base pairs but all nine of the conserved single-stranded residues in the central core are needed for self cleavage. In most cases the mutations did not alter the ability of the hammerhead to assemble into a bimolecular complex. In the few cases where mutant hammerheads did not assemble, it appeared to be the result of the mutation stabilizing an alternate substrate or ribozyme secondary structure. All combinations of mutant substrate and mutant ribozyme were less active than the corresponding single mutations, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA. The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences.

A site-specific self-cleavage reaction performed by a novel RNA in neurospora mitochondria

We describe a novel DNA and RNA found in the mitochondria of the Varkud-1c natural isolate of Neurospora. The majority of the RNA, termed VSRNA, is an 881 nucleotide single-stranded circular molecule complementary to one strand of a low copy, double-stranded circular DNA, VSDNA. VSRNA combines some features of the human hepatitis delta virus, group I introns, retroelements, and plant viral satellite RNAs. VSRNA synthesized in vitro performs a self-cleavage reaction whose products terminate with a 5′ hydroxyl and a 2′,3′ cyclic phosphate. This reaction may be involved in the natural processing pathway of multimeric VSRNA in vivo. VSRNA lacks a hammerhead structure or substantial sequence similarity to any other self-cleaving RNA, suggesting that the RNA structure involved in cleavage may be different from those in previously characterized catalytic RNAs.

Lead-catalyzed cleavage of yeast tRNAPhe mutants

Yeast tRNA(Phe) lacking modified nucleotides undergoes lead-catalyzed cleavage between nucleotides U17 and G18 at a rate very similar to that of its fully modified counterpart. The rates of cleavage for 28 tRNA(Phe) mutants were determined to define the structural requirements of this reaction. The cleavage rate was found to be very dependent on the identity and correct positioning of the two lead-coordinating pyrimidines defined by X-ray crystallography. Nucleotide changes that disrupted the tertiary interactions of tRNAPhe reduced the rate of cleavage even when they were distant from the lead binding pocket. However, nucleotide changes designed to maintain tertiary interactions showed normal rates of cleavage, thereby making the reaction of a useful probe for tRNA(Phe) structure. Certain mutants resulted in the enhancement of cleavage at a "cryptic" site at C48. The sequences of Escherichia coli tRNA(Phe) and yeast tRNA(Arg) were altered such that they acquired the ability to cleave at U17, confirming our understanding of the structural requirements for cleavage. This mutagenic analysis of the lead cleavage domain provides a useful guide for similar analysis of autocatalytic self-cleavage reactions.

Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction

A hammerhead domain of less than 50 nucleotides is responsible for a self-cleavage reaction in the replication of plant RNA pathogens. The hammerhead is composed of three helices joining at a central conserved core of 11 single stranded nucleotides. The core is believed to fold into a tertiary structure that provides functional groups for catalysis and to coordinate one or more divalent metal ions. In this study we use a phosphorothioate substitution interference assay to identify four phosphates in the conserved core which also play a role in the self-cleavage reaction.

A computational approach to the mechanism of self-cleavage of hammerhead RNA.

Extensive minimization and dynamics computational studies of the hammerhead structural domain of the virusoid of lucerne transient streak virus have been carried out. The following observations at the self-cleavage position are derived from the resulting three-dimensional structure: (i) the cytosine base is at the surface and does not interact with another base (it is free to move), and (ii) the ribose-phosphate backbone is forced to take an abrupt turn since it bridges stems I and III, and this turn points the pro-S and pro-R oxygens of the phosphate to the inward side of the hammerhead. These structural features are independent of the hammerhead being open or closed and allow an unencumbered 3'- to 2'-endo conformational change of the ribose with the resultant creation of an unusual stereochemistry that allows a direct in-line self-cleavage reaction. In the closed hammerhead structure, interactions of stems I and II create a vacancy into which the catalytic hydrated Mg(II) may be docked on labile phosphate. This opening is not present if stems I and II are shortened.

Derivation of an infectious viral RNA by autolytic cleavage of in vitro transcribed viral cDNAs.

An in vitro transcription system has been developed that utilizes self-processing to adjust 3' termini in transcribed viral RNAs. Transcription vectors were constructed by ligating a cDNA "cassette" containing self-cleavage sequences from the satellite RNA of tobacco ringspot virus (STobRV RNA) with the 3' end of brome mosaic virus (BMV) cDNA clones. Transcripts were obtained either from templates linearized at positions located downstream from the cassette or from nonlinearized plasmids. In both cases, a spontaneous self-cleavage reaction produced BMV RNAs that contained 19 heterologous nucleotides at their 3' ends. These RNAs were biologically active, indicating that 19 extra nucleotides did not remove the infectivity. The progeny BMV RNA isolated from infected plants did not retain the 19-base heterologous sequence. BMV RNAs containing longer 3' heterologous sequences were not infectious. The STobRV self-cleavage cassette is potentially applicable to a variety of RNA systems.

Proteolytic cleavage of encephalomyocarditis virus capsid region substrates by precursors to the 3C enzyme

Picornavirus protease 3C is normally released from its P3 precursor by two successive self-cleavage reactions. The free enzyme can then catalyze most of the remaining processing events within the viral polyprotein. To investigate the role of the 3C precursors in the processing cascade, we constructed cDNA clones which expressed genetically altered forms of the encephalomyocarditis P3 region in vitro. Site-specific substitutions were introduced into the Gln-Gly residues at the 3B-3C and 3C-3D junctions, and the resulting proteins were tested for their ability to self-process and to catalyze cleavage of viral capsid precursors in cell-free protease assays. We determined that three P3 region precursor proteins (3ABC, 3CD, and P3), harboring inactive cleavage sites, were as active as the free enzyme (3C) in processing assays with capsid substrates. Further, we found that in addition to the naturally occurring Gln-Gly and Gln-Ser amino acid pairs, the encephalomyocarditis 3C enzyme was able to process Gln-Cys but not Gln-Thr, Gln-Ile, Gln-Tyr, Arg-Gly, or Leu-Gly combinations when these residues were substituted into normal cleavage site contexts.

RNA stem stability in the formation of a self-cleaving hammerhead structure

The proposed single-hammerhead structure of the self-cleaving newt RNA is unstable due to a weak stem III and therefore is unable to mediate self-cleavage. A double-hammerhead structure with greater theoretical stability has been shown to mediate the self-cleavage of this RNA (Forster et al., 1988, Nature 334, 265). We have found that the double-hammerhead mediated self-cleavage reaction of a 40 base RNA containing the newt sequence (termed nCG) can be converted to a single-hammerhead reaction by increasing the size of stem III and/or of its loop, thereby enabling a single-hammerhead structure to form. In addition, the 5'-self-cleavage fragment of the nCG RNA can act in trans to mediate the self-cleavage of a full-length RNA by the formation of a partial double-hammerhead structure.

Characterization of self-cleaving RNA sequences on the genome and antigenome of human hepatitis delta virus.

Recently we reported that in vitro RNA transcripts complementary to the genome of hepatitis delta virus (HDV) contain a unique site at which self-cleavage can occur. Subsequent studies showed that a similar self-cleavage site was present on in vitro RNA transcripts of genomic HDV RNA. The same self-cleavage reactions were also found to occur on HDV RNAs from the livers of infected chimpanzees. Using the in vitro RNA it was also possible to determine that the minimum length of contiguous sequence needed for self-cleavage of genomic RNA was 30 bases 5' and 74 bases 3' of the cleavage site. This sequence was not compatible with the "hammerhead" structure hypothesized to be important in the self-cleavage reactions of other RNAs.

Self-cleaving viroid and newt RNAs may only be active as dimers

Avocado sunblotch viroid (ASBV) is a 247-nucleotide, single-stranded, circular RNA. It is considered to replicate via a rolling-circle mechanism in which circular, monomeric plus and minus RNAs act as templates for the synthesis of longer-than-unit-length precursor RNAs. Processing of these RNAs in vivo may occur by a self-cleavage reaction, as indicated by ability of dimeric, linear plus and minus ASBV RNAs to specifically self-cleave in vitro with the excision of a monomeric RNA with 5'-hydroxyl and 2',3'-cyclic phosphodiester termini. A similar self-cleavage reaction has also been reported to occur in an RNA transcript containing a dimeric copy of a tandemly repeated, 330-base-pair sequence of the newt genome. Based on comparisons with self-cleaving plant viral satellite RNAs, hammerhead-shaped active structures, each containing one self-cleavage site, were proposed for the plus and minus ASBV RNAs and the newt RNA, but the stability of these hammerheads has been questioned. Here, more stable active structures that contain two self-cleavage sites are proposed and data supporting these models are presented.

UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA.

The mutation rate of Escherichia coli increases approximately 100-fold after treatment with replication-inhibiting agents such as UV light. This enhanced mutation rate requires the action of the UmuD and UmuC proteins, which are induced as part of the SOS response to DNA damage. To initiate a biochemical characterization of the role of these proteins, we have developed a plasmid system that gives efficient expression of the umuD and umuC genes. The umuD and umuC genes were placed under the control of a regulated phage lambda PL promoter and a synthetic ribosome-binding site, and the distance to the UmuD start was adjusted to maximize gene expression. Starting from this overproduction system, we have purified the UmuD protein and studied its interaction with RecA. The SOS response is turned on by the capacity of RecA protein to mediate cleavage of the LexA repressor for SOS-controlled operons. Others have shown that UmuD exhibits sequence homology to LexA around the cleavage site, suggesting a possible cleavage reaction for UmuD. We show that RecA mediates cleavage of UmuD, probably at this site. As with LexA, UmuD also undergoes a self-cleavage reaction. We infer that RecA-mediated cleavage of UmuD is another role for RecA in SOS mutagenesis, probably activating UmuD for its mutagenic function.