17 Polynucleotide Phosphorylase

Littauer, Uriel Z.; Soreq, Hermona

doi:10.1016/s1874-6047(08)60289-9

Cited by 69 publications

(76 citation statements)

References 219 publications

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“…The multiple bands that were observed upon PNPase binding were due to the mixture of PNPase forms that were present in the purified preparation, as seen in a nondenaturing protein gel (data not shown). Although the active form of PNPase in the cell is a trimer (17), monomer, dimer, and trimer forms could be observed in vitro. For the apparent dissociation constant (K app ) calculations, we assumed that each of these forms bind RNA with approximately the same affinity.…”

Section: Resultsmentioning

confidence: 98%

“…On the other hand, the degradation assay (Fig. 4C) showed that although PNPase could bind trp (A) 17 RNA well, it could not degrade this substrate past the terminator structure that was formed when TRAP was present. A processing product of ϳ142 nt appeared shortly after initiation of the decay reaction, and this product represented a large fraction (60%) of the total full-length RNA.…”

Section: Resultsmentioning

confidence: 99%

“…We considered the possibility that rapid decay of trp leader RNA in vivo was made possible by the addition of a poly(A) tail. A substrate that had 17 A residues at the 3Ј end, termed "trp (A) 17 RNA," was prepared and tested for binding and decay by PNPase. Unlike the case of trp RNA, binding of PNPase to trp (A) 17 RNA was unaffected by the presence of TRAP (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Initiation of decay of Bacillus subtilis trp leader RNA

Deikus

Bechhofer

2007

Journal of Biological Chemistry

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Transcription termination in the leader region of the Bacillus subtilis trp operon is regulated by binding of the 11-mer TRAP complex to nascent trp RNA, which results in formation of a terminator structure. Rapid decay of trp leader RNA, which is required to release the TRAP complex and maintain a sufficient supply of free TRAP, is mediated by polynucleotide phosphorylase (PNPase). Using purified B. subtilis PNPase, we showed that, when TRAP was present, PNPase binding to the 3 end of trp leader RNA and PNPase digestion of trp leader RNA from the 3 end were inefficient. These results suggested that initiation of trp leader RNA may begin with an endonuclease cleavage upstream of the transcription terminator structure. Such cleavage was observed in vivo. Mutagenesis of nucleotides at the cleavage site abolished processing and resulted in a 4-fold increase in trp leader RNA half-life. This is the first mapping of a decay-initiating endonuclease cleavage site on a native B. subtilis RNA.We have identified four 3Ј-to-5Ј exoribonucleases of Bacillus subtilis that can be involved in the decay of mRNA (1). One of these, PNPase, 2 a processive enzyme that degrades RNA phosphorolytically, was shown earlier to be the major exonucleolytic activity in B. subtilis extracts (2), and we have shown more recently that PNPase is the main enzyme responsible for mRNA turnover in vivo (1, 3). A strain with a disruption of the pnpA gene, encoding PNPase, is viable but has a number of phenotypic defects including an inability to regulate transcription of the trp operon (4). In a wild-type strain, the B. subtilis trp operon is regulated at the level of transcription termination (5, 6). When the supply of intracellular tryptophan is low, the trp operon genes are transcribed from a constitutive promoter, and more tryptophan is made. In the presence of ample tryptophan, transcription from this promoter terminates after transcribing the 139-nucleotide (nt) trp leader RNA. Termination is mediated by the trp RNA-binding attenuation protein (TRAP) complex. TRAP is activated by tryptophan itself and binds with high affinity as an 11-mer to 11 trinucleotide repeats located between nt 36 and 91 of the trp leader sequence (Fig. 1B). Binding of the TRAP complex allows a stem-loop transcription terminator structure to form, which consists of nt 108 -133 of the trp leader sequence. In the absence of TRAP binding (i.e. when TRAP is not bound by excess tryptophan), an antiterminator structure forms that precludes formation of the terminator structure, and transcription proceeds into the trp structural genes (Fig. 1A).For the trp system to be regulated by TRAP, sufficient free TRAP complex needs to be present to bind to newly made trp leader RNA, which is constitutively transcribed from the trp promoter. We found recently that the TRAP complex is released from trp leader RNA by rapid degradation of trp leader RNA (4). This degradation is accomplished by PNPase, whereas other 3Ј-to-5Ј exoribonucleases cannot digest RNA that is TRAP-bound, resulting in a...

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Initiation of decay of Bacillus subtilis trp leader RNA

Deikus

Bechhofer

2007

Journal of Biological Chemistry

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“…PNPase (EC 2.7.7.8) was discovered by GrunbergManago and Ochoa while they were studying the mechanism of biological phosphorylation in Azotobacter vinelandii [1] and was characterized by Littauer and Kornberg in studies of the nature of ribonucleotide incorporation into nucleic acids in Escherichia coli [2]. PNPase was the first enzyme to be identified that can catalyze the formation of polynucleotides from ribonucleotides.…”

mentioning

confidence: 99%

“…PNPase catalyzes both processive 3¢ to 5¢ phosphorolysis and 5¢ to 3¢ polymerization of RNA [2][3][4]. In E. coli, PNPase is mostly active in 3¢ to 5¢ phosphorolysis during RNA degradation and 3¢ end processing, but recently a substantial degree of activity in the polymerization of heteropolymeric tails has also been reported [5,6].…”

mentioning

confidence: 99%

The PNPase, exosome and RNA helicases as the building components of evolutionarily-conserved RNA degradation machines

2007

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The structure and function of polynucleotide phosphorylase (PNPase) and the exosome, as well as their associated RNA-helicases proteins, are described in the light of recent studies. The picture raised is of an evolutionarily conserved RNA-degradation machine which exonucleolytically degrades RNA from 3¢ to 5¢. In prokaryotes and in eukaryotic organelles, a trimeric complex of PNPase forms a circular doughnutshaped structure, in which the phosphorolysis catalytic sites are buried inside the barrel-shaped complex, while the RNA binding domains create a pore where RNA enters, reminiscent of the protein degrading complex, the proteasome. In some archaea and in the eukaryotes, several different proteins form a similar circle-shaped complex, the exosome, that is responsible for 3¢ to 5¢ exonucleolytic degradation of RNA as part of the processing, quality control, and general RNA degradation process. Both PNPase in prokaryotes and the exosome in eukaryotes are found in association with protein complexes that notably include RNA helicase.

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Enzymatic RNA synthesis in self‐reproducing vesicles: An approach to the construction of a minimal synthetic cell

Luisi

Walde

Oberholzer

1994

Ber Bunsenges Phys Chem

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Our approach towards “core‐and‐shell self‐reproduction” is reviewed here. With this term, we indicate a process by which the shell‐reproduction of a spherically bounded system (micelles or vesicles) proceeds simultaneously with the replication of nucleic acid which is hosted inside the micelles or vesicles. The realization of such a process is seen as a step towards the construction of a synthetic cell model. Two chemical systems are examined here, one based on the polynucleotide phosphorylase‐catalyzed synthesis of poly(A) starting from ADP; and the other based on the enzyme Qβ replicase, which is able to catalyze the synthesis of a RNA template. In both cases, production of RNA macromolecules proceeds simultaneously with self‐reproduction of the oleic acid/oleate vesicles. One still open question is the determination of the redistribution of the guest macromolecular components during the reproduction of the shell. Within these limits, it is argued that this work offers a system which goes beyond the two approaches to self‐replication presented until now in the literature, namely the template self‐reproduction of linear sequences of oligonucleotides and the autopoietic shell reproduction of micelles and vesicles.

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17 Polynucleotide Phosphorylase

Cited by 69 publications

References 219 publications

Initiation of decay of Bacillus subtilis trp leader RNA

Initiation of decay of Bacillus subtilis trp leader RNA

The PNPase, exosome and RNA helicases as the building components of evolutionarily-conserved RNA degradation machines

Enzymatic RNA synthesis in self‐reproducing vesicles: An approach to the construction of a minimal synthetic cell

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