Identification And Analysis Of Trans-Translation Substrates In Aeromonas Veronii

The trans-translation is one of the most important ribosome rescue systems in the prokaryotic cells.It consists of two parts,including a small protein B(SmpB)and a transfer-massage RNA(tmRNA).When ribosomes stalled,SmpB and tmRNA will bind to the ribosomes,release them and give tag-peptide to the nascent peptides to promote the hydrolysis of them.Those nascent peptides called trans-translation substrates are not random but specific,and they vary in different conditions or species.More and more researches have showed that ribosome stalling is an efficient mechanism for the post-transcriptional regulation in cells,and so trans-translation is also involved in the post-transcriptional regulation of bacterial gene expression.In different bacteria,trans-translation has effects on bacterial growth,virulence,stress response,and other physiological activities,and it also relates to the bacteria tolerance of drugs.Nowadays,the spread of resistant bacteria becomes a huge danger to human health,which leading to consider trans-translation as a potentially attractive target of the new drugs.Our previous experiments have shown that trans-translation was of great importance to the pathogenic bacteria Aeromonas veronii.The loss of trans-translation made Aeromonas veronii grow slower and be more sensitivity to various antibiotics.To further understand the physiological function of trans-translation in Aeromonas veroni cells,the trans-translation substrates were identified from Aeromonas veronii and further studied.The study was consisted of three parts:(1)A tmRNA mutant was constructed,in which the last 6 amino acid codons of the tag-peptide,originally including the hydrolase recognition site,were mutated as six-Histidine tag.They were recalcitrant to degrade when these peptides rescued by trans-translation,and can be easily purified and collected by the Ni+ resin column.Then,the tmRNA mutant was transformed into tmRNA knockout strain of Aeromonas veronii.Growth curves and western blot experiments revealed that the mutated tmRNA acted like the functions of wild type tmRNA to rescue stalled ribosomes,and labeled the trans-translation substrate with a His6 tag.(2)The trans-translation substrates were purified with a Ni+ resin column and identified by mass spectrometry.Compared with wild-type,tmRNA mutant captured totally 16 possible trans-translation substrates.The phage shock protein A(PspA)was selected for further validation,and the results of western blot showed that PspA was indeed rescued by the trans-translation system,and located the labeled site is Ala126 by following identification.(3)The data analysis of mass spectrometry was optimized.By constructing a theoretical database,the protein with tag-peptide in the purified sample could be directly obtained through single mass spectrometry,and the labeled site of the tag-peptide could be located at the same time.The membrane assembly protein AsmA was labeled at 631st amino acid of its encoding region.The mRNA sequence was predicted to form a stem-loop structure in the downstream.It was presumably the key reason that caused the ribosome stalled in the process of translation.According to that,the model of the ribosome stalling caused by AsmA mRNA and following rescue by trans-translation was proposed.This study provided the basis for the further understanding of the trans-translational physiological functions in Aeromonas veronii,and also revealed the relationship between trans-translation and post-transcriptional regulation of gene expression to some extent.