Studies on the Escherichia coli stringent response protein, RelA

Date

2008-09-19T23:47:15Z

Authors

Gao, Saixue

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Abstract

RelA is a guanosine tetraphosphate synthetase which catalyzes the production of (p)ppGpp during the stringent response in Escherichia coli. RelA consists of an N-terminus, which is responsible for the catalytic activity, and a C-terminus, which is thought to be involved in the regulation of RelA activity. Furthermore, the C-terminus has dimerization and ribosome binding ability. ‘RelA-2, which is a fragment of C-terminus, is a major domain responsible for dimerization and ribosome binding. In this study, it was demonstrated that combination of two mutations (C612G, D637R) in ‘RelA-2 significantly reduced the dimerization. This dimerization-defective mutant still bound to ribosomes both in vivo and in vitro, indicating that dimerization is not required for its ribosome binding and that the dimerizaton domain is separated from its ribosome binding domain. The overexpression of the dimerization-defective mutant in amino acid starved cells inhibited chromosome-encoded wild type RelA activity. As a result, the starved cells did not show a stringent response. This finding does not support the oligomerization model proposed by Gropp group. Previous studies in this laboratory have shown, and were confirmed here, that the overexpressed ‘RelA-3, another fragment of C-terminus, which is devoid of dimerization and ribosome binding ability, did not inhibit the RelA activity when cells are under amino acid starvation. This evidence supports the hypothesis that ribosome binding is somehow involved in the regulation of RelA activity. It was demonstrated in this study that RelA was localized to the 50S subunit in vivo by Western Blot analysis. This result confirmed a previous study showing that the 50S subunit had the enzymatic activity in vitro, but not the 30S subunit. However, an in vitro study using pure 50S and 30S ribosomal subunits for the binding experiments indicated that RelA mainly bound to the 30S subunit and weakly to the 50S subunit. A model has been proposed to explain the possible mechanism of ribosome association for RelA. The involvement of L11 and EF-G in the regulation of RelA activity was also investigated. Three residues (C38, G131, and G137) in L11 have been identified to be crucial for the regulation of RelA activity. Three residues (T89, L438, and G628) in EF-G have been identified to be involved in the regulation of RelA activity. These preliminary studies implicate that the regulation of RelA inside amino acid-starved E. coli is complex.

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Keywords

guanosine tetraphosphate synthetase, RelA, ribosome binding, enzymes

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