Abstract
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."
Highlights
The control of the pattern of gene expression is one of the main means by which a prokaryotic or eukaryotic cell reacts to a variation of its environment
It cuts in the middle of GGAG sequences found in early messenger intergenic regions but not GGAG sequences located in coding sequences or in late messengers
Since its discovery in 1988 [7], one of the most intriguing and exciting question concerning the T4-encoded endoribonuclease RegB has been the study of its specificity
Summary
The control of the pattern of gene expression is one of the main means by which a prokaryotic or eukaryotic cell reacts to a variation of its environment. The factors controlling mRNA half-lives are far from being completely understood and are certainly different in prokaryotic and eukaryotic organisms. In the former, in particular, the coupling of transcription and translation events together with the absence of 5Ј 3 3Ј exonucleases impose specific constraints. The importance of RNA-binding proteins in the control of mRNA decay rates is becoming more and more evident It has recently been shown, for example, that the stability of the E. coli ompA mRNA is modulated by its interactions with the host factor I (HfqI) protein, whose concentration depends, in turn, on the growth rate of the bacteria [5]. We could imagine that S1 participates in enzyme specificity by selecting several targets
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