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Interaction studies on bacterial stringent response protein RelA with uncharged tRNA provide evidence for its prerequisite complex for ribosome binding

  • Gajraj Singh KushwahaEmail author
  • Gert Bange
  • Neel Sarovar BhaveshEmail author
Original Article
  • 163 Downloads

Abstract

The bacterial stringent response is regulated by the synthesis of (p)ppGpp which is mediated by RelA in a complex with uncharged tRNA and ribosome. We intended to probe RelA–uncharged tRNA interactions off the ribosome to understand the sequential activation mechanism of RelA. Stringent response is a key regulatory pleiotropic mechanism which allows bacteria to survive in unfavorable conditions. Since the discovery of RelA, it has been believed that it is activated upon binding to ribosomes which already have uncharged tRNA on acceptor site (A-site). However, uncharged tRNA occupied in the A-site of the ribosome prior to RelA binding could not be observed; therefore, recently an alternate model for RelA activation has been proposed in which RelA first binds to uncharged tRNA and then RelA–uncharged tRNA complex is loaded on to the ribosome to synthesize (p)ppGpp. To explore the alternate hypothesis, we report here the in vitro binding of uncharged tRNA to RelA in the absence of ribosome using formaldehyde cross-linking, fluorescence spectroscopy, surface plasmon resonance, size-exclusion chromatography, and hydrogen–deuterium exchange mass spectrometry. Altogether, our results clearly indicate binding between RelA and uncharged tRNA without the involvement of ribosome. Moreover, we have analyzed their binding kinetics and mapping of tRNA-interacting regions of RelA structure. We have also co-purified TGS domain in complex with tRNA to further establish in vivo RelA–tRNA binding. We have observed that TGS domain recognizes all types of uncharged tRNA similar to EF-Tu and tRNA interactions. Altogether, our results demonstrate the complex formation between RelA and uncharged tRNA that may be loaded to the ribosome for (p)ppGpp synthesis.

Keywords

Stringent response RelA ppGpp synthetase tRNA–protein interaction 

Notes

Acknowledgements

This work is funded by Science and Engineering Research Board (SERB), Government of India under Young Scientist Start-up Grant (SB/YS/LS-176/2014) and also supported by EMBO Short-term fellowship to GSK. We thank Mr. Manu Vashist and Advance Instrumentation Research Facility, Jawaharlal Nehru University (JNU), for providing the SPR facility. We thank Wieland Steinchen from the HDX-MS core facility MIDAS (Marburg) for performing the HDX-MS experiments. We thank ICGEB core funds.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

294_2019_966_MOESM1_ESM.docx (240 kb)
Supplementary material 1 (DOCX 240 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Transcription Regulation GroupInternational Centre for Genetic Engineering and Biotechnology (ICGEB)New DelhiIndia
  2. 2.LOEWE Center for Synthetic Microbiology and Department of ChemistryPhilipps UniversityMarburgGermany

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