# Differential Proteomic Analysis of _Listeria monocytogenes_ during High-Pressure Processing
## 4 Discussion
### Translational Regulation  
The ribosome is responsible for protein synthesis, which consists of four essential parts--initiation, elongation, termination, and recycling. Thirty-two ribosomal subunit proteins were found to have significantly upregulated expression, with only one exception, 50S ribosomal protein L33-1, which was downregulated by 4.27 fold in H2/C. However, higher hydrostatic pressure changed the significant downregulation (\(-4.27\) for 200 MPa) of L33-1 to a minor upregulation (0.17 for 400 MPa) (Table S3). Typically, the 32 ribosomal proteins showed a dose-dependent increase in fold change when _L. monocytogenes_ was exposed to pressures of 200 and 400 MPa (Table S3). HPP on ribosome dissociation was shown on vegetative cells of _L. monocytogenes_, _Bacillus subtilis_ and _Escherichia coli_[12; 22; 23]. During the growth arrest phase, HPP induced a significant number (46-58) of upregulated ribosomal protein genes and translation-associated genes, namely translation initiation factors, but merely 3-7 genes were downregulated by more than 2 fold [11]. Sucrose density gradient sedimentation analysis revealed that ribosomes were dissociated in a pressure-dependent manner and then reconstructed. Transcriptome analysis using vegetative cells of _B. subtilis_ also revealed that the translational machinery can preferentially be reconstructed upon the HHP 250 MPa treatment [22]. Furthermore, HPP induced dissociated free 30S and 50S ribosomal subunits, which are more susceptible to degradation by endonucleases into nucleotides than 70S ribosomes, and their ribosome-derived nucleotides could be recycled as a nutrient source for the growth of starved _E. coli_ under nutrient-limiting conditions [24]. Initiation occurs only at the free 30S subunits by interacting with initiation factors (IFs) when a 30S subunit comprises mRNA transcripts, IF-3:IF-1 complex, IF-2, GTP, and a particular initiator, aminoacyl tRNA. The 30S subunits require IF-3 for mRNA binding. Meanwhile, IF-3 has to be released from 30S subunits to create the complex of 50S subunits connected with the mRNA:30S subunit. As a result, initiation is the rate-limiting step in protein synthesis, and therefore several critical translational regulations occur during initiation. This study showed that all IFs increased under the HPP 200 and 400 MPa treatments, although both the IF-1 and IF-2 levels slightly increased. Moreover, IF-3 increased by 4.08 fold under H4/C treatment (Table 1). The increases in IFs and ribosomal subunit proteins indicated that the HPP at 200 and 400 MPa treatments might promote the initiation of protein synthesis. The essential composite for peptide chain elongation is an mRNA:70S ribosome:peptide/tRNA complex, aminoacyl tRNA, elongation factors, and GTP. A significant increase in IF-3 may occupy 30S subunits to prevent re-associating with 50S subunits and hinder the subsequent elongation process. tRNA is a vital adaptor molecule in protein synthesis. Specific aminoacyl-tRNA synthetases (aaRSs) produce aminoacyl tRNA by conjugating amino acids to the 3\({}^{\prime}\)-CAA acceptor end of tRNA. Each aminoacyl-tRNA synthetase matches up its amino acid, with tRNA having proper anticodons that can base-pair with codons from mRNA specifying the particular amino acid. The correct loading of amino acids onto various tRNA is crucial to achieving translation fidelity. The aaRSs serve a dual role in charging tRNAs, providing protein synthesis flux and reducing uncharged tRNA levels. ThrRS, responsible for aminoacylation of threnoyl-tRNA, was downregulated by \(-1.08\) log\({}_{2}\) fold in H4/C, but not significant (\(-0.64\)) in H2/C (Table S3). Moreover, AsnRS, ArgRS, IleRS, TyrRS, AspRS, ProRS, and CysRS were all downregulated but not significant in both the H2/C and H4/C conditions. The three proteins, GaC/A/B, develop correctly charged Asn-tRNA or Gln-tRNA through the transamidation of misacylated Asp-tRNA or Glu-tRNA in prokaryotes which are commonly lacking either or both asparaginyl-tRNA and glutaminyl-tRNA synthetases.