Molecular dissection of the mycobacterial stringent response protein Rel.
Latency in Mycobacterium tuberculosis poses a barrier in its complete eradication. Overexpression of certain genes is one of the factors that help these bacilli survive inside the host during latency. Among these genes, rel, which leads to the expression of Rel protein, plays an important role by synthesizing the signaling molecule ppGpp using GDP and ATP as substrates, thereby changing bacterial physiology. In Gram-negative bacteria, the protein is thought to be activated in vivo in the presence of ribosome by sensing uncharged tRNA. In the present report, we show that Rel protein from Mycobacterium smegmatis, which is highly homologous to M. tuberculosis Rel, is functional even in the absence of ribosome and uncharged tRNA. From the experiments presented here, it appears that the activity of Rel(Msm) is regulated by the domains present at the C terminus, as the deletion of these domains results in higher synthesis activity, with little change in hydrolysis of ppGpp. However, in the presence of tRNA, though the synthesis activity of the full-length protein increases to a certain extent, the hydrolysis activity undergoes drastic reduction. Full-length Rel undergoes multimerization involving interchain disulfide bonds. The synthesis of pppGpp by the full-length protein is enhanced in the reduced environment in vitro, whereas the hydrolysis activity does not change significantly. Mutations of cysteines to serines result in monomerization with a simultaneous increase in the synthesis activity. Finally, it has been possible to identify the unique cysteine, of six present in Rel, required for tRNA-mediated synthesis of ppGpp.
Jain
Vikas
V
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Saleem-Batcha
Raspudin
R
China
Arnab
A
Chatterji
Dipankar
D
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics; D030562:Databases, Protein; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006868:Hydrolysis; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics
D006153:Guanosine Diphosphate; D012694:Serine; D012343:RNA, Transfer; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases; D003545:Cysteine