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At least 19 recordsLinked to original sources
PubMed1998

Spermine switches a Neurospora VS ribozyme from slow Cis cleavage to fast trans cleavage.

In keeping with the known role of polyamines as counterions in RNA folding, we have found that concentrations of spermine as low as 1 microM facilitate first-order Cis cleavage and decrease the concentration of magnesium required for optimal cleavage of the VS ribozyme. Surprisingly, under certain experimental conditions, cleavage reactions at concentrations of spermine above about 20 microM were not first-order. At 100 microM spermine, about half of the RNA cleaved in an initial very fast burst, k >/= 5 min-1, about 100-fold faster than under our previously optimized conditions; the remainder of the RNA cleaved more slowly. The extent of the burst and the initial rate of cleavage were proportional to RNA concentration, suggesting that the fast phase was due to intermolecular trans cleavage involving two or more RNAs. This inference of trans cleavage was confirmed by demonstrating spermine-dependent trans cleavage of particular combinations of mutant RNAs that were each incapable of cis cleavage. The experimental conditions required to switch the VS ribozyme into trans cleavage mode are quite stringent. The RNA must be preincubated with an adequate concentration of spermine at very low ionic strength near neutral pH. Concentrations of buffers and salts typically used in in vitro studies of ribozymes, including those used in our previous characterization of the VS ribozyme, are sufficiently high that they prevent or reverse the trans cleaving RNA conformation. The ability to switch cleavage modes from cis to trans provides an experimental system to study different active conformations of VS RNA, as well as to investigate the functional consequences of polyamine-RNA interactions.

Base Sequence↗
PubMed2002

Efficient trans-cleavage by the Schistosoma mansoni SMalpha1 hammerhead ribozyme in the extreme thermophile Thermus thermophilus.

The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo. The cellular role of these repetitive elements and their transcripts is unknown. Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo. We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMalpha DNA family from the human parasite Schistosoma mansoni. The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme-product dissociation step. The optimal temperature for trans-cleavage was 70 degrees C. This result was confirmed when both the SMalpha1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus. Moreover, SMalpha1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMalpha1 RNA has been selected for stability. Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo.

Animals↗
PubMed1995

In vivo and in vitro trans-cleavage activity of hepatitis C virus serine proteinase expressed by recombinant baculoviruses.

By the use of recombinant baculoviruses, the trans-cleavage of hepatitis C virus (HCV) non-structural polyprotein was studied. The viral serine proteinase encoded by the NS3 gene was expressed efficiently in insect cells infected with a baculovirus recombined with HCV cDNA corresponding to amino acids 1046-1243 and the signal sequence of the rabies virus G protein. Coinfection studies showed the in vivo trans-cleavage activity of the expressed protein by the use of a recombinant producing NS5 as a substrate. We also found that the partially purified NS3 serine proteinase prepared from the recombinant-infected cells could cleave NS5A/5B substrate. Characterization of the proteinase obtained wil provide basic knowledge on processing of the HCV polyprotein.

Amino Acid Sequence↗
PubMed2000

Intracellular ribozyme-catalyzed trans-cleavage of RNA monitored by fluorescence resonance energy transfer.

Small catalytic RNAs like the hairpin ribozyme are proving to be useful intracellular tools; however, most attempts to demonstrate trans-cleavage of RNA by ribozymes in cells have been frustrated by rapid cellular degradation of the cleavage products. Here, we describe a fluorescence resonance energy transfer (FRET) assay that directly monitors cleavage of target RNA in tissue-culture cells. An oligoribonucleotide substrate was modified to inhibit cellular ribonuclease degradation without interfering with ribozyme cleavage, and donor (fluorescein) and acceptor (tetramethylrhodamine) fluorophores were introduced at positions flanking the cleavage site. In simple buffers, the intact substrate produces a strong FRET signal that is lost upon cleavage, resulting in a red-to-green shift in dominant fluorescence emission. Hairpin ribozyme and fluorescent substrate were microinjected into murine fibroblasts under conditions in which substrate cleavage can occur only inside the cell. A strong FRET signal was observed by fluorescence microscopy when substrate was injected, but rapid decay of the FRET signal occurred when an active, cognate ribozyme was introduced with the substrate. No acceleration in cleavage rates was observed in control experiments utilizing a noncleavable substrate, inactive ribozyme, or an active ribozyme with altered substrate specificity. Subsequently, the fluorescent substrates were injected into clonal cell lines that expressed cognate or noncognate ribozymes. A decrease in FRET signal was observed only when substrate was microinjected into cells expressing its cognate ribozyme. These results demonstrate trans-cleavage of RNA within mammalian cells, and provide an experimental basis for quantitative analysis of ribozyme activity and specificity within the cell.

Animals↗
PubMed1994

Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics.

The hepatitis C virus H strain (HCV-H) polyprotein is cleaved to produce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. An HCV-encoded serine proteinase activity in NS3 is required for cleavage at four sites in the nonstructural region (3/4A, 4A/4B, 4B/5A, and 5A/5B). In this report, the HCV-H serine proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-5B substrate with an inactivated serine proteinase domain, trans-cleavage was observed at all sites except for the 3/4A site. Deletion of the inactive proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were processed efficiently in trans; however, cleavage of an NS4B-5A substrate occurred only when the serine proteinase domain was coexpressed with NS4A. Only the N-terminal 35 amino acids of NS4A were required for this activity. Thus, while NS4A appears to be absolutely required for trans-cleavage at the 4B/5A site, it is not an essential cofactor for serine proteinase activity. To begin to examine the conservation (or divergence) of serine proteinase-substrate interactions during HCV evolution, we demonstrated that similar trans-processing occurred when the proteinase domains and substrates were derived from two different HCV subtypes. These results are encouraging for the development of broadly effective HCV serine proteinase inhibitors as antiviral agents. Finally, the kinetics of processing in the nonstructural region was examined by pulse-chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and significant quantities of various polyprotein intermediates were observed. NS5B, the putative RNA polymerase, was found to be significantly less stable than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression of other viral polypeptides, including the HCV-encoded proteinases.

Amino Acid Sequence↗
PubMed2000

Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated cell lines.

A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3'-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent trans-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic amino-terminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C.

Animals↗
PubMed2001

Exploring the minimal substrate requirements for trans-cleavage by RNase P holoenzymes from Escherichia coli and Bacillus subtilis.

We analysed the processing of small bipartite model substrates by Escherichia coli and Bacillus subtilis RNase P and corresponding hybrid enzymes. We demonstrate specific trans-cleavage of a model substrate with a 4 bp stem and a 1 nucleotide (nt) 5' flank, representing to date the smallest mimic of a natural RNase P substrate that could be processed in trans at the canonical RNase P cleavage site. Processing efficiencies decreased up to 5000-fold when the 5' flank was shortened from 3 to 1 nt. Reduction of the 5' flank to 1 nt was more deleterious than reducing the stem from 7 to 4 bp, although the 4 bp duplex formed only transiently, in contrast to the stable 7 bp duplex. These results indicate that the crucial contribution of nt -2 in the single-stranded 5' flank to productive interaction is a general feature of A- and B-type bacterial RNase P enzymes. We also showed that an Rp-phosphorothioate modification at nt -2 interferes with processing. Bacterial RNase P holoenzymes are also capable of cleaving single-stranded RNA oligonucleotides as short as 5 nt, yielding RNase P-specific 5'-phosphate and 3'-OH termini, with measured turnover rates of up to 0.7 min-1. All cleavage sites were at least 2 nt away from the 5' and 3' ends of the oligonucleotides. Some cleavage site preferences were observed dependent on the identity of the RNase P RNA subunit.

Bacillus subtilis↗
PubMed1991

Efficient trans cleavage and a common structural motif for the ribozymes of the human hepatitis delta agent.

Cis-active ribozymes are potential therapeutic agents; however, to be used in this capacity, they must first be converted to trans-active ribozymes, a process facilitated by analysis of their structures. We present evidence that the genomic and antigenomic ribozymes of the human delta hepatitis agent share a structural ("axehead") motif that has conserved sequence elements and a stable hairpin. Guided by the features of the axehead, we divided each of the delta ribozymes into two subdomains, which we synthesized as separate RNA transcripts to give an enzyme and substrate for each ribozyme. Incubation of a substrate subdomain with its matching enzyme resulted in efficient and accurate trans cleavage. This work forms the basis for kinetic studies and for adapting the delta ribozymes for cleavage of selected target RNAs.

Base Sequence↗
PubMed1995

Efficient trans-cleavage of a stem-loop RNA substrate by a ribozyme derived from neurospora VS RNA.

We have constructed a ribozyme containing 144 nucleotides of Neurospora VS RNA that can catalyze the cleavage of a separate RNA in a true enzymatic manner (Km approximately 0.13 microM, kcat approximately 0.7/min). Comparison of the rates of cis- and trans-cleavage, as well as the lack of effect of pH on the rate of cleavage, suggest that a rate-limiting step, possibly a conformational change, occurs prior to cleavage. The minimum contiguous substrate sequence required for cleavage consists of one nucleotide upstream and 19 nucleotides downstream of the cleavage site. Unlike most other ribozymes which interact with long single-stranded regions of their substrates, the minimal substrate for the VS ribozyme consists mostly of a stable stem-loop, which would appear to preclude its recognition simply via extensive Watson-Crick base pairing.

Base Sequence↗
PubMed1991

cis- and trans-cleavage activities of poliovirus 2A protease expressed in Escherichia coli.

The poliovirus protease, 2Apro, was produced in Escherichia coli from plasmids that encode a fusion protein consisting of the N-terminal portion of the bacterial TrpE protein linked to poliovirus 2Apro. This fusion protein underwent efficient autocatalytic cleavage at the N terminus of 2Apro, generating the mature protease. Extracts of bacteria expressing 2Apro induced the specific cleavage of the p220 subunit of the eukaryotic translation initiation factor 4F, similar to the 2Apro-mediated reaction that occurs in poliovirus-infected HeLa cells. A portion of the poliovirus polyprotein containing the 2Apro cleavage site at the P1/P2 junction was produced by translation of cDNA transcripts in rabbit reticulocyte lysates and then tested as a substrate for 2Apro-mediated cleavage. The protein was partially cleaved by 2Apro in trans. Finally, a 16-amino-acid synthetic peptide, representing the P1/P2 junction sequence, was analyzed as a substrate for 2Apro. The peptide was labeled with fluorescein at a lysine residue to facilitate its detection. Recombinant 2Apro cleaved the synthetic peptide into two half-peptide molecules which were resolved by high-pressure liquid chromatography. Direct sequence analysis of the isolated peptide products demonstrated that cleavage occurred at the expected tyrosine-glycine pair. A rapid cleavage assay for 2Apro activity on the synthetic peptide was developed, using separation of the fluorescein-labeled 8-amino-acid product from the 16-residue substrate by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels.

Base Sequence↗
PubMed1994

Identification of the domain required for trans-cleavage activity of hepatitis C viral serine proteinase.

A serine proteinase, Cpro-2, encoded in the hepatitis C virus (HCV) genome, is considered to be located in the N-terminal part of HCV p70, one of the putative nonstructural (NS) proteins of HCV. Cpro-2 is suggested to be responsible for producing several kinds of NS proteins by processing of the HCV precursor polyprotein. We identified the active domain of Cpro-2 and clarified the mechanism of HCV polyprotein processing; various HCV mutants deleted around this serine proteinase structure were cosynthesized with unprocessed HCV polypeptides containing Cpro-2-dependent cleavage sites in COS-1 cells. We showed that Cpro-2 cleaved the HCV precursor polyprotein intermolecularly (trans) and that Cpro-2 domain which is necessary and sufficient for that cleavage mapped to within 167 aa, from Gly1049 to Ser1215 of the HCV precursor polyprotein.

Animals↗
PubMed1993

Different target-site specificities of the hairpin ribozyme in cis and trans cleavages.

The hairpin ribozyme cleaves a phosphodiester bond at the 5' side of a 5'GUC3' sequence of an RNA with high efficiency. An RNA having a 5'GUA3' sequence instead of the GUC sequence is a poor substrate for this ribozyme. Here, we show that this is indeed so in a trans-acting ribozyme system, but in a cis-acting ribozyme system this ribozyme cleaves the 5' side of a GUA sequence as efficiently as the wild-type cleaves the GUC sequence. One base substitution in the ribozyme also affected the target-site specificity in the cis-acting system.

Base Sequence↗
PubMed1999

High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme.

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product.

Base Sequence↗
PubMed1993

Mutational analysis of the amino acid residues essential for the cis and trans cleavage activity of the potato virus Y 50-kDa protease.

To examine the proteolytic activities of various truncated derivatives of the potato virus Y (PVY) 50-kDa protease, the derivatives were expressed in Escherichia coli in polyprotein forms fused with coat protein (CP). For the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-CP junction. The activity was evaluated by the amount of the mature CP released from the precursor by the intra- and intermolecular cleavage occurring in E. coli. By this experiment, we identified the moiety responsible for the proteolytic activity of the 50-kDa protease to be a 26-kDa polypeptide mapped to the C-terminal half of the protease. Introduction of His234-->Tyr, Asp269-->Asn, or Cys339-->Gly substitution in the putative catalytic triad of the protease abolished its activity. However, the mutated protease with Cys339-->Ser replacement retained a reduced proteolytic activity.

Amino Acid Sequence↗
PubMed1992

DNA cleavage in trans by the active site tyrosine during Flp recombination: switching protein partners before exchanging strands.

Each recombination event mediated by the Flp recombinase is the sum of four strand breakage and reunion reactions executed in two steps of two-strand exchanges. The reaction requires four Flp monomers. The key catalytic residue in Flp is Tyr-343. Arg-191, His-305, and Arg-308 appear to facilitate the cleavage and exchange steps of recombination. These four residues constitute the invariant tetrad of the Int family site-specific recombinases. Complementation tests between "step-arrest" mutants of Flp suggest that each Flp protomer harbors a "fractional active site." Hybrid "half site-recombinase" complexes reveal that efficient catalysis occurs when the Arg-His-Arg triad is present on one Flp monomer and the active site Tyr on a second monomer. Strand cleavage by an Flp monomer occurs virtually exclusively on the half site to which its partner protein is bound (cleavage in trans), and almost never on the half site to which it is bound (cleavage in cis). Trans-cleavage by Flp can provide a means for functionally exchanging Flp monomers between two DNA partners. Such a mechanism would be germane to recombination, since cleavage and rejoining in cis can only restore the parental substrate configuration and cannot yield recombinants.

Amino Acid Sequence↗
PubMed2000

Mutational analysis of the NIa protease from pepper vein banding potyvirus.

The nuclear inclusion protein a (NIa) protease plays an important role in the life cycle of potyviruses by processing the viral polyprotein into functional proteins. For functional characterization, the NIa protease from Pepper vein banding potyvir s (PVBV) was overexpressed in Escherichia coli and purified. Using a recombinant polyprotein substrate containing the nuclear inclusion protein b (NIb)-coat protein (CP) cleavage site, a trans-cleavage assay was developed for the NIa protease. The polyprotein substrate also possessed the cleavage site between NIa and NIb, in addition to the NIb-CP site. However, no trans-cleavage by the NIa protease between NIa and NIb was detected indicating that the cleavage between NIa and NIb under natural conditions would be by a cis-cleavage reaction. Site-specific mutations of the conserved residues D81, D90, C110, T146, C151 and H167 were performed to investigate their roles in the catalytic process of the protease. Such an analysis has revealed that D81 and C151 constitute two of the catalytic triad residues in the NIa protease, D90 and C110 are not essential for catalysis, and T146 and H167 are probably involved in binding to Gln at the P1 position of the substrate.

Capsid↗
PubMed1991

Identification of essential amino acid residues in the functional activity of poliovirus 2A protease.

Proteolytic processing of poliovirus polyprotein is carried out by the products of two viral genes, 2A and 3C. 2A protease catalyzes cleavage of the polyprotein of type 1 poliovirus at two sites, one a cis cleavage at the 2A N-terminus and the other a trans cleavage within the 3D polymerase. In addition to polyprotein cleavage activity, 2A protease also indirectly induces cleavage of the p220 component of the cap-binding protein complex, which results in selective inhibition of host protein synthesis. Molecular genetic and biochemical analyses of 2A protease were performed to test its putative homology to small trypsin-like serine proteases and to examine the roles of individual amino acids in the reaction mechanism of 2A protease. A recombinant plasmid containing poliovirus 1C, 1D, and 2A gene sequences was expressed in a cell-free transcription/translation system, resulting in synthesis of a precursor protein that underwent efficient self-processing and produced mature 2A protease. To identify residues involved in the catalytic center and/or substrate-binding loops, we generated a series of 2A mutants by site-specific mutagenesis of this plasmid. Mutants were then expressed in vitro and tested for autocatalytic cis cleavage activity, trans cleavage of the 1D/2A junction, and trans-activation of p220-specific protease. Our data suggest that the conserved His20, Asp38, and Cys109 residues recently proposed to be equivalent to the catalytic triad of known serine proteases may comprise the catalytic triad of 2A protease. Surprisingly, Asp38 could be replaced with glutamic acid and retain autocatalytic function. Other amino acid substitutions at Tyr88, Tyr89, and Thr124 suggested that these residues lie in loops involved in substrate binding. Biochemical studies with protease inhibitors indicate that 2A protease activity is blocked by inhibitors specific for serine and cysteine proteases. Overall, the results are consistent with the hypothesis that 2A proteinase is structurally similar to the trypsin-like family of serine proteases with the substitution of cysteine 109 as the active site nucleophile.

Amino Acid Sequence↗