The bacteriophage T4 regB ribonuclease. Stimulation of the purified enzyme by ribosomal protein S1.
Infection of Escherichia coli by bacteriophage T4 induces a mRNA ribonuclease activity that shows specificity for cleavage within the sequence GGAG. Substrates of the activity in vivo include a number of phage mRNAs that are cleaved at GGAGs within the Shine/Dalgarno domains of their translation initiation regions. Induction of the ribonuclease depends on the product of the T4 gene regB. We describe here the overproduction and extensive purification of the RegB protein. RegB precisely co-purifies with an activity that cleaves within the sequence GGAG in oligonucleotide and polynucleotide RNAs and is therefore likely to constitute the sequence-specific catalytic component of the observed activity. We further report that the low cleavage rate observed with our preparations of purified RegB is substantially increased (1-2 orders of magnitude) by the addition of E. coli ribosomal protein S1. We discuss the implications of this observation for the mechanism of action of the RegB ribonuclease in vitro and in vivo.
Ruckman
J
J
Molecular, Cellular, and Developmental Biology Department, University of Colorado, Boulder 80309.
Ringquist
S
S
Brody
E
E
Gold
L
L
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005814:Genes, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012260:Ribonucleases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D012260:Ribonucleases