Kinetic characterization of ribozymes directed against the cisplatin resistance-associated ABC transporter cMOAT/MRP2/ABCC2.
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The enhanced expression of the human ABC transporter, cMOAT (MRP2/ABCC2), is associated with resistance of tumor cells against platinum-containing compounds, such as cisplatin. Therefore, cMOAT represents an interesting candidate factor for modulation of antineoplastic drug resistance. Two different hammerhead ribozymes, which exhibit high catalytic cleavage activities towards specific RNA sequences encoding cMOAT, were designed. Cleavage sites of these ribozymes are the GUC sites in codons 704 and 708 of the open reading frame in the cMOAT-specific mRNA molecule. Hammerhead ribozymes were in vitro synthesized using bacteriophage T7 RNA polymerase and oligonucleotide primers whereby one primer contains a T7 RNA polymerase promoter sequence. cMOAT-encoding substrate RNA molecules were created by a reverse transcription polymerase chain reaction using RNA prepared from the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS overexpressing the cMOAT-encoding transcript. In a cell-free system, both anti-cMOAT ribozymes cleaved their substrate in a highly efficient manner at a physiologic pH and temperature. The cleavage reaction was dependent on time and ribozyme:substrate ratio for determining specific kinetic parameters. (+info)
Selection and characterization of a high-activity ribozyme directed against the antineoplastic drug resistance-associated ABC transporter BCRP/MXR/ABCG2.
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Breast cancer resistance protein (BCRP) is a recently identified new member of the superfamily of ATP-binding cassette transporters. BCRP is a "half transporter" that may homo- or heterodimerize to form an active transport complex. A considerable overexpression of BCRP was reported from various atypical multidrug-resistant tumor cell lines, in particular from those which were established by treatment with mitoxantrone. Thus, BCRP represents a very interesting candidate molecule for reversal of a drug-resistant phenotype. Six hammerhead ribozymes directed against the BCRP-encoding mRNA were designed and tested for their ability to cleave their target molecule. The anti-BCRP ribozymes were in vitro synthesized using bacteriophage T7 RNA polymerase and oligonucleotide primers whereby one primer contains a T7 RNA polymerase promoter sequence. BCRP-encoding substrate RNA molecules were created by a reverse transcription polymerase chain reaction using total RNA prepared from the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV exhibiting a high BCRP mRNA expression level. One anti-BCRP ribozyme was found to show a very high endoribonucleolytic cleavage activity at physiologic pH and temperature. This ribozyme was characterized in a cell-free system with regard to its specific kinetic parameters using large target molecules. (+info)
Specific recognition of protein carboxy-terminal sequences by natural IgM antibodies in normal serum.
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Our previous study indicated that normal serum contains complement-fixing natural IgM antibodies reacting with a large variety of randomly generated protein carboxy-termini. Here we show that the "carboxy-terminal" IgM (C-IgM) antibodies specifically react with short peptide sequences located immediately at the protein carboxy-terminus. The specificity of C-IgM-peptide interactions is tentatively defined by three to four amino acid residues. All carboxy-terminal peptides in a large peptide library apparently react with C-IgM antibodies. Immobilized synthetic peptides also react with C-IgM antibodies. No interaction of C-IgM antibodies with internal peptide sequences has been observed. C-IgM antibodies are present in germ-free and in athymic adult rats and are absent in newborn rats. The natural ubiquity of protein carboxy-termini in biological structures suggests that C-IgM could play an important role in antigen clearance and presentation to the immune system. From a practical viewpoint, the recognition of carboxy-terminal peptides by complement-fixing C-IgM antibodies has profound implications for the use of peptide- and protein-derivatized delivery vehicles and artificial materials. (+info)
Structure in nascent RNA leads to termination of slippage transcription by T7 RNA polymerase.
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T7 RNA polymerase presents a very simple model system for the study of fundamental aspects of transcription. Some time ago it was observed that in the presence of only GTP as a substrate, on a template encoding the initial sequence GGGA., T7 RNA polymerase will synthesize a 'ladder' of poly-G RNA products. At each step, the ratio of elongation to product release is consistently approximately 0.75 until the RNA reaches a length of approximately 13-14 nt, at which point this ratio drops precipitously. One model to explain this drop in complex stability suggests that the nascent RNA may be structurally hindered by the protein; the RNA may be exiting via a pathway not taken by normally synthesized RNA and therefore becomes sterically destabilized. The fact that the length of RNA at which this occurs is close to the length at which the transition to a stably elongating complex occurs might have led to other mechanistic proposals. Here we show instead that elongation falls off due to the cooperative formation of structure in the nascent RNA, most likely an intramolecular G-quartet structure. Replacement of GTP by 7-deaza-GTP completely abolishes this transition and G-ladder synthesis continues with a constant efficiency of elongation beyond the limit of detection. The polymerase-DNA complex creates no barrier to the growth of the nascent (slippage) RNA, rather termination is similar to that which occurs in rho-independent termination. (+info)
Polymerase slippage at vesicular stomatitis virus gene junctions to generate poly(A) is regulated by the upstream 3'-AUAC-5' tetranucleotide: implications for the mechanism of transcription termination.
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Termination of mRNA synthesis in vesicular stomatitis virus (VSV), the prototypic rhabdovirus, is controlled by a 13-nucleotide gene end sequence which comprises the conserved tetranucleotide 3'-AUAC-5', the U(7) tract and the intergenic dinucleotide. mRNAs terminated at this sequence possess 100- to 300-nucleotide-long 3' poly(A) tails which are thought to result from polymerase slippage (reiterative transcription) by the VSV polymerase on the U(7) tract. Previously we determined that in addition to the AUAC tetranucleotide, the U(7) tract was an essential signal in the termination process. Shortening or interrupting the U(7) tract abolished termination. These altered U tracts also prevented the polymerase from performing reiterative transcription necessary for generation of the mRNA poly(A) tail and thus established seven residues as the minimum length of U tract that allowed reiterative transcription to occur. In this study we investigated whether sequences other than the essential U(7) tract are involved in controlling polymerase slippage. We investigated whether the AUAC tetranucleotide affected the process of reiterative transcription by analyzing the nucleotide sequence of RNAs transcribed from altered subgenomic templates and infectious VSV variants. The tetranucleotide was found to regulate reiterative transcription on the U(7) tract. The extent of polymerase slippage was governed not by specific tetranucleotide sequences but rather by nucleotide composition such that slippage occurred when the tetranucleotide was composed of A or U residues but not when it was composed of G or C residues. This suggested that polymerase slippage was controlled, at least in part, by the strength of base pairing between the template and nascent strands. Further data presented here indicate that the tetranucleotide contains both a signal that directs the VSV polymerase to slip on the downstream U(7) tract and also a signal that directs a slipping polymerase to terminate mRNA synthesis. (+info)
Tuning DNA "strings": modulating the rate of DNA replication with mechanical tension.
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Recent experiments have measured the rate of replication of DNA catalyzed by a single enzyme moving along a stretched template strand. The dependence on tension was interpreted as evidence that T7 and related DNA polymerases convert two (n = 2) or more single-stranded template bases to double helix geometry in the polymerization site during each catalytic cycle. However, we find structural data on the T7 enzyme--template complex indicate n = 1. We also present a model for the "tuning" of replication rate by mechanical tension. This model considers only local interactions in the neighborhood of the enzyme, unlike previous models that use stretching curves for the entire polymer chain. Our results, with n = 1, reconcile force-dependent replication rate studies with structural data on DNA polymerase complexes. (+info)
Characterisation of the structure of ocr, the gene 0.3 protein of bacteriophage T7.
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The product of gene 0.3 of bacteriophage T7, ocr, is a potent inhibitor of type I DNA restriction and modification enzymes. We have used biophysical methods to examine the mass, stability, shape and surface charge distribution of ocr. Ocr is a dimeric protein with hydrodynamic behaviour equivalent to a prolate ellipsoid of axial ratio 4.3 +/- 0.7:1 and mass of 27 kDa. The protein is resistant to denaturation but removal of the C-terminal region reduces stability substantially. Six amino acids, N4, D25, N43, D62, S68 and W94, are all located on the surface of the protein and N4 and S68 are also located at the interface between the two 116 amino acid monomers. Negatively charged amino acid side chains surround W94 but these side chains are not part of the highly acidic C-terminus after W94. Ocr is able to displace a short DNA duplex from the binding site of a type I enzyme with a dissociation constant of the order of 100 pM or better. These results suggest that ocr is of a suitable size and shape to effectively block the DNA binding site of a type I enzyme and has a large negatively charged patch on its surface. This charge distribution may be complementary to the charge distribution within the DNA binding site of type I DNA restriction and modification enzymes. (+info)
Role of the C-terminal residue of the DNA polymerase of bacteriophage T7.
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The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites. (+info)