Synthesis and hydrolysis of pppGpp in mycobacteria: a ligand mediated conformational switch in Rel.
Bacteria respond to starvation by synthesizing a polyphosphate derivative of guanosine, (p)ppGpp, that helps the bacteria in surviving during stress. The protein in Gram-positive organisms required for (p)ppGpp synthesis is Rel, a bifunctional enzyme that carries out both synthesis and hydrolysis of this molecule. Rel shows increased pppGpp synthesis in the presence of uncharged tRNA, the effect of which is regulated by the C-terminal of Rel. We show by fluorescence resonance energy transfer that the distance between the N-terminus cysteine residue at the catalytic domain and C692 at the C-terminus increases upon the addition of uncharged tRNA. In apparent anomaly, the steady state anisotropy of the Rel protein decreases upon tRNA binding suggesting "compact conformation" vis-à-vis "open conformation" of the free Rel. We propose that the interaction between C692 and the residues present in the pppGpp synthesis site results in the regulated activity and this interaction is abrogated upon addition of uncharged tRNA. We also report here the binding of pppGpp to the C-terminal part of the protein that leads to more unfolding in this region.
Jain
Vikas
V
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Saleem-Batcha
Raspudin
R
Chatterji
Dipankar
D
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D025461:Feedback, Physiological; D006158:Guanosine Pentaphosphate / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008024:Ligands; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism
D008024:Ligands; D006158:Guanosine Pentaphosphate; D012343:RNA, Transfer; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases; D003545:Cysteine