The enzyme from bacteriophage T4 cleaves early mRNAs between Ap and Gp at one specific specific GpGpApGp site, favouring their further transition to middle-phase mRNA. The activity is enhanced by Ribosomal S1 protein. The enzyme catalyses a two-stage endonucleolytic cleavage. The first reaction produces 5'-hydroxy-phosphooligonucletides and 3'-phosphooligonucleotides ending with 2',3'-cyclic phosphodiester, which are released from the enzyme. The enzyme then hydrolyses these cyclic compounds in a second reaction that takes place only when all the susceptible 3',5'-phosphodiester bonds have been cyclised. The second reaction is a reversal of the first reaction using the hydroxyl group of water instead of the 5'-hydroxyl group of ribose. The overall process is that of a phosphorus-oxygen lyase followed by hydrolysis to form the 3'-nucleotides.
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SYSTEMATIC NAME
IUBMB Comments
[pre-mRNA]-guanosine-adenosine 5'-hydroxy-guanosine-ribonucleotide-3'-[RNA fragment]-lyase (cyclicizing; [RNA fragment]-3'- adenosine -2',3'-cyclophosphate-forming and hydrolysing)
The enzyme from bacteriophage T4 cleaves early mRNAs between Ap and Gp at one specific specific GpGpApGp site, favouring their further transition to middle-phase mRNA. The activity is enhanced by Ribosomal S1 protein. The enzyme catalyses a two-stage endonucleolytic cleavage. The first reaction produces 5'-hydroxy-phosphooligonucletides and 3'-phosphooligonucleotides ending with 2',3'-cyclic phosphodiester, which are released from the enzyme. The enzyme then hydrolyses these cyclic compounds in a second reaction that takes place only when all the susceptible 3',5'-phosphodiester bonds have been cyclised. The second reaction is a reversal of the first reaction using the hydroxyl group of water instead of the 5'-hydroxyl group of ribose. The overall process is that of a phosphorus-oxygen lyase followed by hydrolysis to form the 3'-nucleotides.
the enzyme cleaves with an almost absolute specificity its RNA substrate in the middle of the GGAG tetranucleotide mainly found in the Shine-Dalgarno sequence
the sequence-specific endoribonuelease RegB introduces cuts in early phage messenger RNAs. In most cases, cutting takes place in the middle of the tetranacleotide GGAG. Efficient cleavages occur in the motifs located in intergenic regions, some of them being Shine-Dalgamo sequences. When located in a coding sequence, this tetranucleotide is poorly recognized or not at all
the enzyme is necessary for the degradation of early but not middle or late mRNAs. The synthesis of several early proteins is down-regulated, probably as a consequence of RegB cleavages in the Shine-Dalgarno sequence of these genes. The synthesis of several other proteins is up-regulated, suggesting that processing by RegB might improve translation by changing the conformation of a transcript
the enzyme participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. It specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites
the enzyme cleaves early mRNAs between Ap and Gp at one specific specific GpGpApGp site. Many intergenic GGAG motifs, including Shine-Dalgarno sequences, are not substrates for RegB
the enzyme is necessary for the degradation of early but not middle or late mRNAs. The synthesis of several early proteins is down-regulated, probably as a consequence of RegB cleavages in the Shine-Dalgarno sequence of these genes. The synthesis of several other proteins is up-regulated, suggesting that processing by RegB might improve translation by changing the conformation of a transcript
the enzyme participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. It specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites
the enzyme is necessary for the degradation of early but not middle or late mRNAs. The synthesis of several early proteins is down-regulated, probably as a consequence of RegB cleavages in the Shine-Dalgarno sequence of these genes. TRegB also regulates the translation of several prereplicative genes
inactivates a sub-class of early mRNA by cleaving Shine-Dalgamo sequences, and (ii) it is necessary for the degradation of early mRNAs, but not of middle and late mRNAs
mutant enzyme binds its RNA substrate with the same affinity as the wild type protein. The beta-sheet content of the RegB H48A secondary structure is probably poor but not nil
1.2 mM samples of RegB H48A precipitate at 32.5°C within 18 h. In the presence of sodium citrate molecules no visible precipitation after a 3 days-incubation
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
by coupling the toxic restriction endoribonuclease RegB, from the bacteriophage T4, to the prokaryotic T7 and the eukaryotic GAL1 promoters, a two-function plasmid called pTOXR-1 is constructed. This plasmid is a zero-background cloning vector. It allows an efficient positive selection of recombinant plasmids without the need to completely digest, dephosphorylate, or purify the vector prior to the ligation step. The pTOXR-1 positive selection system requires no special Escherichia coli strains, no special culture media, and no addition of inducer to the selective plates. In addition, since this vector carries all signals required for both prokaryotic and eukaryotic expression, it allows the overproduction of heterologous proteins, fused to a polyhistidine tag, in the bacterium Escherichia coli and in the yeast Saccharomyces cerevisiae from a single plasmid. Hence, this vector may be a useful time-saving tool for onestep cloning and versatile protein expression in bacteria and yeast
expression in Escherichia coli strain BL21(DE3)-pLysS, transformed with plasmid pEH48A. RegB is highly toxic to Escherichia coli, preventing its overproduction from standard expression vectors
the wild-type RegB protein and the four histidine-to-alanine mutants are expressed in Escherichia coli strain XL1-Blue using the phage lCE6 induction system
First structural investigation of the restriction ribonuclease RegB NMR spectroscopic conditions, 13C/15N double-isotopic labelling and two-dimensional heteronuclear spectra