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The stringent response modulators, guanosine tetraphosphate (ppGpp) and protein DksA, bind RNA polymerase (RNAP) and regulate gene expression to adapt bacteria to different environmental conditions. Here, we use Atomic Force Microscopy and in vitro transcription assays to study the effects of these modulators on the conformation and stability of the open promoter complex (RPo) formed at the rrnA P1, rrnB P1, its discriminator (dis) variant and λ pR promoters. In the absence of modulators, RPo formed at these promoters show different extents of DNA wrapping which correlate with the position of UP elements. Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner. Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site. At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role.

Phosphoglycerate mutase (PGAM) activity in promastigotes of the protozoan parasite Leishmania mexicana is found only in the cytosol. It corresponds to a cofactor-independent PGAM as it is not stimulated by 2,3-bisphosphoglycerate and is susceptible to EDTA and resistant to vanadate. We have cloned and sequenced the gene and developed a convenient bacterial expression system and a high-yield purification protocol. Kinetic properties of the bacterially produced protein have been determined (3-phosphoglycerate:The activity is inhibited by phosphate but is resistant to Cland SO 4 2-. Inactivation by EDTA is almost fully reversed by incubation with CoCl 2 but not with MnCl 2 , FeSO 4 , CuSO 4 , NiCl 2 or ZnCl 2 . Alkylation by diethyl pyrocarbonate resulted in irreversible inhibition, but saturating concentrations of substrate provided full protection. Kinetics of the inhibitory reaction showed the modification of a new group of essential residues only after removal of metal ions by EDTA. The modified residues were identified by MS analysis of peptides generated by trypsin digestion. Two substrate-protected histidines in the proximity of the active site were identified (His136, His467) and, unexpectedly, also a distant one (His160), suggesting a conformational change in its environment. Partial protection of His467 was observed by the addition of 25 lM CoCl 2 to the EDTA treated enzyme but not of 125 lM MnCl 2 , suggesting that the latter metal ion cannot be accommodated in the active site of Leishmania PGAM.Keywords: chemical modification; kinetics; Leishmania mexicana; metal dependence; phosphoglycerate mutase.The reversible isomerization of 2-phosphoglycerate (2PGA) and 3-phosphoglycerate (3PGA) is an obligate step for both glycolysis and gluconeogenesis. This step is carried out in two different ways in nature, by two different types of evolutionarily unrelated enzymes (although both EC 5.4.2.1). The better documented enzyme is the cofactor-dependent phosphoglycerate mutase (d-PGAM) due to its requirement for 2,3-bisphophoglycerate. It is present in some eubacteria, yeast and all vertebrates most frequently as a dimer or tetramer of 23-30-kDa subunits [1]. The second enzyme, called cofactor-independent phosphoglycerate mutase (i-PGAM), is a monomeric protein of 60 kDa. Upon comparative sequence and structure analysis, the former enzyme has been classified as a member of the phospho-histidine acid phosphatase superfamily [2] and the latter as a member of the metal-dependent alkaline phosphatase superfamily [3,4]. Whereas d-PGAM is the enzyme present in all vertebrates, i-PGAM is found in all plants and archaebacteria [5] and, together with d-PGAMs, in lower eukaryotes and eubacteria [1,6].We have previously shown that an i-PGAM participates in glycolysis in the protist Trypanosoma brucei [7], a human pathogen. The completely distinct structures and catalytic mechanisms of trypanosomal and human PGAM offer great promise for the design of inhibitors with high selectivity for the parasite's enzyme. Therefore, this fi...

Knots are natural topologies of chains. Yet, little is known about spontaneous knot formation in a polypeptide chain—an event that can potentially impair its folding—and about the effect of a knot on the stability and folding kinetics of a protein. Here we used optical tweezers to show that the free energy cost to form a trefoil knot in the denatured state of a polypeptide chain of 120 residues is 5.8 ± 1 kcal mol−1. Monte Carlo dynamics of random chains predict this value, indicating that the free energy cost of knot formation is of entropic origin. This cost is predicted to remain above 3 kcal mol−1 for denatured proteins as large as 900 residues. Therefore, we conclude that naturally knotted proteins cannot attain their knot randomly in the unfolded state but must pay the cost of knotting through contacts along their folding landscape.