While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.

Cellular and Molecular Microbiology Faculté des Sciences Université libre de Bruxelles Campus La Plaine Building BC Boulevard du Triomphe 1050 Brussels Belgium

Department of Clinical Microbiology Rigshospitalet 2200 Copenhagen Denmark

Department of Experimental Medical Science Lund University 221 00 Lund Sweden

Deutsches Elektronen Synchrotron DESY Centre for Structural Systems Biology Notkestr 85 22607 Hamburg Germany

Faculty of Life Sciences Kyoto Sangyo University Kamigamo Motoyama Kita ku Kyoto 603 8555 Japan

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nám 2 166 10 Prague 6 Czech Republic

Laboratory for Molecular Infection Medicine Sweden Umeå University 901 87 Umeå Sweden

University of Tartu Institute of Technology 50411 Tartu Estonia

WELBIO Avenue Hippocrate 75 1200 Brussels Belgium

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