Preferential degradation of polyadenylated and polyuridinylated RNAs by the bacterial exoribonuclease polynucleotide phosphorylase.
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Polyadenylation of mRNA has been shown to target the RNA molecule for rapid exonucleolytic degradation in bacteria. To elucidate the molecular mechanism governing this effect, we determined whether the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated PNPase degraded polyadenylated and non-polyadenylated RNAs at similar rates. However, when the two molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. The same phenomenon was observed with polyuridinylated RNA. The poly(A) tail has to be located at the 3' end of the RNA, as the addition of several other nucleotides at the 3' end prevented competition for polyadenylated RNA. In RNA-binding experiments, E. coli PNPase bound to poly(A) and poly(U) sequences with much higher affinity than to poly(C) and poly(G). This high binding affinity defines poly(A) and poly(U) RNAs as preferential substrates for this enzyme. The high affinity of PNPase for polyadenylated RNA molecules may be part of the molecular mechanism by which polyadenylated RNA is preferentially degraded in bacterial cells. (+info)
Kinetic and product distribution analysis of human eosinophil cationic protein indicates a subsite arrangement that favors exonuclease-type activity.
(10/527)
With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up)nU>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonuclease-like mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1 sites, located upstream of the P-O-5' cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism. (+info)
Aspirochlorine: a highly selective and potent inhibitor of fungal protein synthesis.
(11/527)
Aspirochlorine, a compound belonging to the gliotoxin family of compounds, exhibits antifungal and antibacterial activity but its mechanism of action remains unknown. In this study we show that aspirochlorine inhibits the pathogenic fungus Candida albicans by acting on fungal protein synthesis. The compound selectively inhibits cell-free protein synthesis when using a C. albicans system, but does not inhibit this synthesis in vitro when tested with bacterial and mammalian systems. Moreover, in intact C. albicans cells, aspirochlorine inhibits protein synthesis but does not inhibit chitin, DNA or glucan synthesis though at high concentrations some inhibition of RNA synthesis is observed. By contrast, in intact Bacillus subtilis cells, aspirochlorine did not inhibit protein, DNA, or cell wall synthesis though it significantly inhibited RNA synthesis. Furthermore, using heterologous systems (mammalian ribosomes and C. albicans cytosolic factors) the data suggest that the inhibitory action of aspirochlorine is not exerted through a direct interaction with C. albicans EF-1 or EF-2. (+info)
Mapping of the RNA-binding and endoribonuclease domains of NIPP1, a nuclear targeting subunit of protein phosphatase 1.
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NIPP1 (351 residues) is a major regulatory and RNA-anchoring subunit of protein phosphatase 1 in the nucleus. Using recombinant and synthetic fragments of NIPP1, the RNA-binding domain was mapped to the C-terminal residues 330-351. A synthetic peptide encompassing this sequence equalled intact NIPP1 in RNA-binding affinity and could be used to dissociate NIPP1 from the nuclear particulate fraction. An NIPP1 fragment consisting of residues 225-351 (Ard1/NIPP1gamma), that may be encoded by an alternatively spliced transcript in transformed B-lymphocytes, displayed a single-strand Mg(2+)-dependent endoribonuclease activity. However, full-length NIPP1 and NIPP1(143-351) were not able to cleave RNA, indicating that the endoribonuclease activity of NIPP1 is restrained by its central domain. The endoribonuclease activity was also recovered in the RNA-binding domain, NIPP1(330-351), but with a 30-fold lower specific activity. Thus, the endoribonuclease catalytic site and the RNA-binding site both reside in the C-terminal 22 residues of NIPP1. The latter domain does not conform to any known nucleic-acid binding motif. (+info)
Effects of oligonucleotide length and atomic composition on stimulation of the ATPase activity of translation initiation factor elF4A.
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Eukaryotic translation initiation factor 4A (elF4A) has been proposed to use the energy of ATP hydrolysis to remove RNA structure in the 5' untranslated region (UTR) of mRNAs, helping the 43S ribosomal complex bind to an mRNA and scan to find the 5'-most AUG initiator codon. We have examined the effect of changing the atomic composition and length of single-stranded oligonucleotides on binding to elF4A and on stimulation of its ATPase activity once bound. Substitution of 2'-OH groups with 2'-H or 2'-OCH3 groups reduces ATPase stimulation at least 100-fold, to background levels, without significantly affecting oligonucleotide affinity. These effects suggest that 2'-OH groups participate in an elF4A conformational change that occurs subsequent to oligonucleotide binding and is required for ATPase stimulation. Replacing nonbridging oxygen atoms in phosphodiester linkages with sulfur atoms to make phosphorothioate linkages has no significant effect on stimulation, while substantially increasing affinity. Extending the length of an RNA oligonucleotide from 4 to approximately 15 nt gradually increases oligonucleotide affinity and ATPase stimulation. Consistent with this observation, the increase in affinity and stimulation provided by phosphorothioate linkages and 2'-OH groups is proportional to the number of these groups present within larger oligonucleotides. Further, changing the position of blocks of phosphorothioate linkages or 2'-OH groups within a larger oligonucleotide does not affect affinity and has only a small effect on stimulation. These observations suggest that numerous interactions between the oligonucleotide and elF4A contribute individually to binding and ATPase stimulation. Nevertheless, significant stimulation is observed with as few as four RNA residues. These properties may allow elF4A to operate within regions of 5' UTRs containing only short stretches of exposed single-stranded RNA. As stimulation increases when longer stretches of single-stranded RNA are available, it is possible that the accessibility of single-stranded RNA in a 5' UTR influences translation efficiency. (+info)
Human hepatic glyceraldehyde-3-phosphate dehydrogenase binds to the poly(U) tract of the 3' non-coding region of hepatitis C virus genomic RNA.
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The unique poly(U/UC) tract, the middle part of the tripartite 3' non-coding region (3'NCR) of hepatitis C virus (HCV) genomic RNA, may represent a recognition signal for the HCV replicase complex. In this study, several proteins binding specifically to immobilized ribooligonucleotide r(U)(25) mimicking this structure were identified using cytosolic extracts from HCV-negative or -positive liver explants, and a prominent 36 kDa protein was studied further. Competition experiments including homoribopolymers revealed binding affinities in the order: oligo/poly(U)>(A)>(C)>(G). The protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multifunctional protein known to bind RNA. GAPDH bound efficiently to the full-length HCV RNA and binding to various 3'NCR constructs revealed critical dependence upon the presence of the middle part of the 3'NCR. Polypyrimidine tract-binding protein, described previously to bind the 3'NCR, did not bind efficiently to the middle part of 3'NCR and was captured from liver extracts in considerably smaller quantities. (+info)
Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules.
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Single molecules of DNA or RNA can be detected as they are driven through an alpha-hemolysin channel by an applied electric field. During translocation, nucleotides within the polynucleotide must pass through the channel pore in sequential, single-file order because the limiting diameter of the pore can accommodate only one strand of DNA or RNA at a time. Here we demonstrate that this nanopore behaves as a detector that can rapidly discriminate between pyrimidine and purine segments along an RNA molecule. Nanopore detection and characterization of single molecules represent a new method for directly reading information encoded in linear polymers, and are critical first steps toward direct sequencing of individual DNA and RNA molecules. (+info)
Polyuridylated mRNA synthesized by a recombinant influenza virus is defective in nuclear export.
(16/527)
The poly(A) tail of influenza virus mRNA is synthesized by reiterative copying of a U track near the 5' end of the virion RNA (vRNA) template by the viral RNA polymerase. We have engineered a novel influenza A/WSN/33 virus which contains a neuraminidase (NA) vRNA with its U track mutated into an A track. Instead of synthesizing poly(A)-tailed NA mRNA, this novel virus synthesizes poly(U)-tailed NA mRNA. In infected cells, most poly(U)-tailed NA mRNA was retained in the nucleus, while most control polyadenylated NA mRNA was transported to the cytoplasm. These results suggest that the poly(A) tail is important for efficient nuclear export of NA mRNA. The mutant virus produced a reduced amount of NA and showed an attenuated phenotype, suggesting that poly(A) signal mutants of this type might be useful as potential live attenuated virus vaccines. In addition, this virus mutant might provide a useful model to further elucidate the basic mechanisms of mRNA nuclear export. (+info)