Fig. 1.

mRNA is a potent inhibitor of ppGpp synthesis by E. faecalis RelQ. (A) ³H ppGpp synthesis activity of 250 nM E. faecalis RelQ (62.5 nM tetrameric RelQ) in the presence (gray bars) and absence (empty bars) of 100 μM ppGpp, as well as starved ribosomal complexes or individual components thereof. Note that ppGpp is a strong activator of RelQ’s enzymatic activity and mitigates the inhibition by starved ribosomal complexes or mRNA(MF). (B and C) Single-stranded mRNA inhibits RelQ’s activity in a sequence-specific manner, and this inhibition is countered by ppGpp. Titrations were performed with increasing concentrations of either single-stranded (empty circles) or double-stranded (filled circles) RNA, in the absence (black circles) or presence (red circles) of 100 μM ppGpp. All reaction mixtures contained 250 nM (62.5 nM tetramer) E. faecalis RelQ, 300 μM ³H GDP, and 1 mM ATP. Titration data were fitted with the 4PL model. Error bars represent SDs of the turnover estimates determined by linear regression. Each experiment was performed at least three times.