# Differential Proteomic Analysis of _Listeria monocytogenes_ during High-Pressure Processing
## 4 Discussion
### HPP Promoted Translation Initiation and Retarded Ribosome Biogenesis  
The maturation factors, including ribosome maturation factor M (RimM), 30S ribosome-binding factor A (RbfA), ribosomal RNA small subunit dimethyl transferase A (Ksga/RsmA), and GTPase (Era), are responsible for ribosomal biogenesis. Era can bind to 16S rRNA to prevent 30S and 50S joining, facilitating rRNA maturation and 30S ribosome biogenesis. RsmA, ribosome biogenesis factor, binds to a pre-30S particle and specifically dimethylates two conserved adenoses in the terminal of 16S rRNA helix [26]. RbfA is associated with 16S rRNA but not 70S ribosomes or polysomes, and is essential in late-30S ribosomal subunit maturation [27]. Recently, RbfA and RimM were found to be involved in two distinct 30S assembly stages consisting of the early development of the central pseudoknot with the folding of the head and further docking of helix 44 to form important inter-subunit bridges in the mature structure at a later stage [28]. A comprehensive review revealed that increased RbfA synthesis could moderately attenuate \(\triangle rimM\) mutation. The overexpression of Era and RsmA proteins can repress \(\triangle rbfA\) mutation and cold-sensitive _Era_ mutant, respectively. This implied a functional order for the four proteins, RimM, RbfA, Era, and RsmA, with RsmA as perhaps the final protein to complete 30S ribosome assembly in bacteria [29]. This study showed that RimM (\(-\)0.70 in H2/C and \(-\)1.09 in H4/C), RbfA (\(-\)4.10 in H2/C and \(-\)0.87 in H4/C), Era (\(-\)0.34 in H2/C and \(-\)0.46 in H4/C), and RsmA (\(-\)0.64 in H2/C and \(-\)0.88 in H4/C) were all reduced under the 200 and 400 MPa treatments, although the result was only significant for RimM in H4/C, and RbfA in H2/C (Table S3). The decreases in ribosomal maturation factors indicated that HPP at 200 and 400 MPa might hinder ribosome maturation and biogenesis. The latest study showed that RbfA in the immature _E. coli_ 30S subunit could promote ribosome biogenesis and suppress protein synthesis; instead, RbfA is displaced by IF-3 after 30S maturation initiates translation [29]. In this study, IF-3 was markedly upregulated, and RbfA was downregulated. This might indicate that abundant IF-3 could substitute RbfA to bind with 30S ribosome, facilitating translation initiation for better adaption to nutrition starvation, extreme temperature, and antibiotic stress. Hence, loss of RbfA and elevated IF-3 during HPP treatments might maintain the fidelity of bacterial protein synthesis for better tolerability to HPP treatments. Moreover, the shortage of charged aminoacyl tRNA stringently induces the accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA, activating the production of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (ppPGPP) to terminate rRNA transcription and suspend ribosome assembly [30]. This study revealed that aaRSs comprising AlaRS, LysRS, ThrRS, IleRS, ArgRS, TyrrRS, ProRS, AsnRS, CysRS and AspRS, as well as the GaTC/A/B system, were all reduced in H4/C, although the findings were significant only for ThrRS in H4/C and GaTC in H2/C and H4/C (Table 1 and Table S3), implying that HPP inhibits ribosome assembly. HPP induced ribosome dissociation, resulting in the abundant ribosomal subunit protein, and thus accelerated feedback regulation to hinder ribosome biogenesis [11; 30]. This study showed that 32 ribosomal subunit proteins were significantly increased (Figure 1, Table S3), indicating that HPP may moderate ribosome biogenesis. Finally, the rRNA operon (\(rrn\)) anti-termination system is vital in rRNA synthesis, folding, and ribosome activity. Nus proteins can topologically constrain the 165 rRNA in a growing loop that supports standard rRNA folding and processing. The Nus-modified transcription complex in rrn operons serves as an RNA chaperone for assisting 165 rRNA folding and RNase III processing, resulting in the formation of functional 30S ribosome subunits [31].