# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly
## 2 A Brief Overview of Ribosome Assembly  
The _E. coli_ 70S ribosome is a 2.3 MDa complex with a diameter of 210 A [25]. Up to 70,000 70S ribosomes are estimated to be present within a single _E. coli_ cell, constituting between 40%-50% of the cellular dry weight when dividing [26; 27]. The ribosome is a ribozyme consisting of roughly 1/3 protein and 2/3 rRNA, which can be further broken down into the 30S small and 50S large subunits. The 30S ribosome is responsible for binding and decoding the mRNA by recognising the complementary base interactions between the mRNA codon and the transfer RNA (tRNA) anticodon [28]. The 50S subunit houses the peptidyltransferase centre (PTC) machinery within domain V of the 23S rRNA and confers amino acid residues onto the novel peptide chain [28]. The PTC is made up of an aminoacyl site (A-site), peptidyltransferase site (P-site) and an exit site (E-site). In addition, the 50S can also prevent premature peptide hydrolysis and the exit tunnel quaternary conformation can aid in the co-translational folding of the nascent chain [29]. The assembly of prokaryotic ribosomes involves the coordinated action of a number of steps, including: rRNA transcription, with subsequent modifications such as pseudouridylation and methylation; r-protein transcription, translation and modification; correct folding and association of both rRNA with r-proteins and the binding and dissociation of biogenesis cofactors. This is thought to proceed via two major strategies: co-transcriptional assembly and rRNA folding-guided assembly [30]. Electron microscopy images revealed the near immediate association of constitutively expressed r-protein complexes to nascent rRNA chains upon transcription by RNA Polymerase (RNAP) [31]. The second strategy suggests that ribosome assembly is limited by RNA folding and that upon correct secondary conformation the relevant r-proteins can associate and stabilise the RNA [32; 33]. This folding of rRNA can occur either in a protein-dependent or protein-independent manner [32], mediated by assembly factors which often use GTPase activity as a mechanism of dissociation from the ribosome following correct maturation (reviewed extensively in [5; 34; 35; 36; 37; 38; 39]). In agreement with this, the 30S subunit is thought to assemble from the 5'-3' of the 16S rRNA, in line with the orientation of transcription and subsequent folding [40]. While the assembly of r-proteins onto the immature subunits exhibits some modularity [2], the correct processing and protection of the rRNA is essential for the functionality of the ribosome. During the first step of the ribosome assembly pathway, the three rRNAs are cotranscribed into one precursor transcript from _rrn_ operons (Figure 1). Different bacterial species contain differing numbers of _rrn_ operons, which comprise up to 1% of the genome. For instance, _E. coli_ contains seven operons, while _Bacillus subtilis_ contains 10. Once transcribed, the precursor rRNA is subject to ribonuclease (RNase) III processing into the 17S, 25S, 5S and tRNA (Figure 1) [41]. Subsequent secondary structure formation of the immature 17S rRNA recruits associated ribosomal proteins during 30S formation, with several hierarchies of proteins present (reviewed by [38]). The primary r-proteins bind directly to the rRNA, secondary protein binding is reliant on primary protein presence and, lastly, tertiary protein binding to the secondary r-proteins occurs [1], with each binding event stabilising the rRNA fold [40]. 17S processing in _E. coli_ is thought to occur on the ribosome itself, with rapid removal of 115 nucleotides from the 5' by RNases E and G and 33 nucleotides from the 3' via the activity of several RNases including the endoribonuclease YbeY and the redundant 3'-5' exoribonucleases RNase R, PH, II and polynucleotide phosphorylase (Figure 1) [42; 43; 44]. The ribonucleases involved in rRNA maturation differ drastically between species, especially when comparing Proteobacteria and Firmicutes.