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 e arly messengers. It cuts specifically in the middle of GGAG sequences found in early messenger intergenic regions but not GGAG sequences located in co ding sequences or in late messengers. In vitro RegB activity is very low bu t is enhanced by a factor up to 100 by the ribosomal protein S1. In the abs ence of clear sequence motif distinguishing substrate and non-substrate GGA G-containing RNAs, we postulated the existence of a structural determinant. To test this hypothesis, we correlated the structure, probed by NMR spectr oscopy, 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 SI. In the absence of S1, RegB efficiently hydr olyses substrates in which the last G of the GGAG motif is located in a sho rt stem between two loops. Both strengthening and weakening of this structu re strongly decrease the cleavage rate, indicating that this structure cons titutes a positive cleavage determinant. Based on our results and those of others, we speculate that S1 favors the formation of the structure recogniz ed by RegB and can thus be considered a "presentation protein".