Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However, tRNA-saturation curves, performed at two different ribosome/stringent factor ratios and filter-binding assays, did not support this hypothesis.
The stringent response plays a significant role in the survival of bacteria during different environmental conditions. It is activated by the binding of stringent factor (SF) to stalled ribosomes that have an unacylated tRNA in the ribosomal A-site which leads to the synthesis of (p)ppGpp. ppGpp binds to the RNA polymerase, resulting in a rapid down-regulation of rRNA and tRNA transcription and up-regulation of mRNAs coding for enzymes involved in amino acid biosynthesis. The importance of the A-site and unacylated tRNA in the activation of SF was confirmed by chemical modification and subsequent primer extension experiments (footprinting experiments) which showed that binding of SF to ribosomes resulted in the protection of regions in 23S rRNA, the A-loop and helix 89 that are involved in the binding of the A-site tRNA. An in vitro assay showed that the ribosomal protein L11 and its flexible N-terminal part was important in the activation of SF. Interestingly the N-terminal part of L11 was shown to activate SF on its own and this activation was dependent on both ribosomes and an unacylated tRNA in the A-site. The N-terminal part of L11 was suggested to mediate an interaction between ribosome-bound SF and the unacylated tRNA in the A-site or interact with SF and the unacylated tRNA independently of each other. Footprinting experiments showed that SF bound to the ribosome protected bases in the L11 binding domain of the ribosome that were not involved in an interaction with ribosomal protein L11. The sarcin/ricin loop, in close contact with the L11 binding domain on the ribosome and essential for the binding and activation of translation elongation factors was also found to be protected by the binding of SF. Altogether the presented results suggest that SF binds to the factor-binding stalk of the ribosome and that activation of SF is dependent on the flexible N-terminal domain of L11 and an interaction of SF with the unacylated tRNA in the A-site of the 50S subunit.