# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly  
[PERSON]  
1 The Florey Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2 TN, UK; [EMAIL_ADDRESS] (D.J.B. ); seirving1@sheffield. subclass are the TRAFAC (Translation Factor Association) GTPases, associated with all aspects of translation, cellular motility, intracellular transport and signal transduction [4]. TRAFAC GTPases are more functionally diverse than the SIMIBI family and many fall under the umbrella of RA-GTPases--a term used to refer to any GTPase involved in ribosome assembly, translation or any other ribosomal process. These GTPases are reported to have an affinity for GTP in the mid-micromolar range [5], although RA-GTPases with much higher affinities for GTP have been identified [6], whereas many Ras-like GTPases have affinities within the low micromolar to picomolar range [5]. This is significant as the physiological concentration of GTP in the bacterial cytoplasm is around 200-500 \(\upmu\)M [7], thus enabling RA-GTPases to respond to small fluctuations in the cellular energy state in order to regulate energy-intensive processes such as ribosome assembly and translation. Many TRAFAC RA-GTPases are important for the correctly timed assembly of both early and late-stage r-proteins onto the mature rRNA [8]. Often considered a checkpoint of accurate ribosome assembly, the purpose of these GTPases is to bind a specific, immature section of the ribosome precursor in the GTP-bound state, hence preventing the association of downstream r-proteins prior to the correct conformation of the RA-GTPase binding site [8]. Maturation of the section in question then stimulates the enzymatic GTPase activity and subsequent dissociation of the RA-GTPase from the ribosome, removing the steric hindrance and allowing further subunit assembly [5]. During conditions of intense conditional stress, the transcriptome and proteome of bacteria are altered to facilitate survival using a signalling pathway termed the stringent response. This pathway was first discovered through investigation of the response of _E. coli_ to amino acid starvation. The starved _E. coli_ cells produced two nucleotides that appeared as'magic spots' on a chromatograph and were later identified as the nucleotides ppGpp and ppPGpp, which function as the effectors of cellular change [9]. Across bacteria, amino acid starvation appears to be a common trigger of the stringent response because amino acids are universally required for growth. However, it is also activated by additional key stressors, such as antibiotics, iron limitation, immature tRNAs and fatty acid limitation [10; 11; 12; 13; 14]. There are three groups of proteins responsible for the synthesis and hydrolysis of (p)ppGpp in the cell: small alarmone synthetases (SASs), small alarmone hydrolases (SAHs) and long RelA/SpoT homologues (long-RSHs) [15; 16]. These groups are members of the RelA/SpoT homologues (RSH) superfamily, named after the two (p)ppGpp synthesis enzymes found in _E. coli_, RelA and SpoT [15]. ppGpp and ppPGpp are synthesised by the transfer of a pyrophosphate group from ATP to the ribose 3'-OH of GDP or GTP, respectively, by nucleophilic substitution [17; 18]. Hydrolysis of (p)ppGpp releases the pyrophosphate group and either GDP or GTP. The stringent response is characterised by an accumulation of up to 2 mM (p)ppGpp, which results in a general reduction in transcription, particularly in genes involved in biosynthesis of macromolecules [19]. Translation is also reduced during the stringent response as a result of an inhibition at multiple points along the protein production route, including a reduction in rRNA transcription, impaired ribosome maturation and an inhibition of translation from mature ribosomes [6; 20; 21; 22].