Positive allosteric feedback regulation of the stringent response enzyme RelA by its product.

During the stringent response, Escherichia coli enzyme RelA produces the ppGpp alarmone, which in turn regulates transcription, translation and replication. We show that ppGpp dramatically increases the turnover rate of its own ribosome-dependent synthesis by RelA, resulting in direct positive regulation of an enzyme by its product. Positive allosteric regulation therefore constitutes a new mechanism of enzyme activation. By integrating the output of individual RelA molecules and ppGpp degradation pathways, this regulatory circuit contributes to a fast and coordinated transition to stringency.

Thermodynamic characterization of ppGpp binding to EF-G or IF2 and of initiator tRNA binding to free IF2 in the presence of GDP, GTP, or ppGpp.

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 µM K(d)versus 9.1-13.9 µM K(d) at 10-25 °C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA(fMet) demonstrating that IF2 binds to initiator tRNA with submicromolar K(d) and that affinity is altered by the G nucleotides only slightly. This--in conjunction with earlier reports on IF2 interactions with fMet-tRNA(fMet) in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA(fMet) binding and GTP was suggested to strongly promote fMet-tRNA(fMet) binding--sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA(fMet) selection.

Thermodynamics of GTP and GDP binding to bacterial initiation factor 2 suggests two types of structural transitions.

During initiation of messenger RNA translation in bacteria, the GTPase initiation factor (IF) 2 plays major roles in the assembly of the preinitiation 30S complex and its docking to the 50S ribosomal subunit leading to the 70S initiation complex, ready to form the first peptide bond in a nascent protein. Rapid and accurate initiation of bacterial protein synthesis is driven by conformational changes in IF2, induced by GDP-GTP exchange and GTP hydrolysis. We have used isothermal titration calorimetry and linear extrapolation to characterize the thermodynamics of the binding of GDP and GTP to free IF2 in the temperature interval 4-37 degrees C. IF2 binds with about 20-fold and 2-fold higher affinity for GDP than for GTP at 4 and 37 degrees C, respectively. The binding of IF2 to both GTP and GDP is characterized by a large heat capacity change (-868+/-25 and -577+/-23 cal mol(-1) K(-1), respectively), associated with compensatory changes in binding entropy and enthalpy. From our data, we propose that GTP binding to IF2 leads to protection of hydrophobic amino acid residues from solvent by the locking of switch I and switch II loops to the gamma-phosphate of GTP, as in the case of elongation factor G. From the large heat capacity change (also upon GDP binding) not seen in the case of elongation factor G, we propose the existence of yet another type of conformational change in IF2, which is induced by GDP and GTP alike. Also, this transition is likely to protect hydrophobic groups from solvent, and its functional relevance is discussed.