Role of the substrate conformation and of the S1 protein in the cleavage efficiency of the T4 endoribonuclease RegB.
The T4 endoribonuclease RegB is involved in the inactivation of the phage early messengers. It cuts specifically in the middle of GGAG sequences found in early messenger intergenic regions but not GGAG sequences located in coding sequences or in late messengers. In vitro RegB activity is very low but is enhanced by a factor up to 100 by the ribosomal protein S1. In the absence of clear sequence motif distinguishing substrate and non-substrate GGAG-containing RNAs, we postulated the existence of a structural determinant. To test this hypothesis, we correlated the structure, probed by NMR spectroscopy, with the cleavage propensity of short RNA molecules derived from an artificial substrate. A kinetic analysis of the cleavage was performed in the presence and absence of S1. In the absence of S1, RegB efficiently hydrolyses substrates in which the last G of the GGAG motif is located in a short stem between two loops. Both strengthening and weakening of this structure strongly decrease the cleavage rate, indicating that this structure constitutes a positive cleavage determinant. Based on our results and those of others, we speculate that S1 favors the formation of the structure recognized by RegB and can thus be considered a "presentation protein."
Lebars
I
I
Groupe de Resonance Magnétique Nucléaire, Laboratoire Département de Chimie, Synthèse Organique, Ecole Polytechnique, 91128 Palaiseau, France.
Hu
R M
RM
Lallemand
J Y
JY
Uzan
M
M
Bontems
F
F
D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology; D007438:Introns; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism
D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D014764:Viral Proteins; C015494:ribosomal protein S1