Effect Of E.coli MRNA Peripheral Localization On Its Degradation Rate

The balance between mRNA transcription and degradation is an effective factor to regulate mRNA abundance and gene expression,and is of great significance for the implementation of normal physiological functions of living organisms.mRNA degradation in eukaryotic and prokaryotic cells includes:endonuclease degradation,5'?3'direction exonuclease degradation and 3'?5'direction exonuclease degradation.The most important degradation pathway in bacteria is endonuclease degradation.Ribonuclease E?Ribonuclease E,RNase E?is an important endo-ribonuclease involved in RNA post-transcription processing and mRNA degradation,and is a key component of E.coli mRNA degradation pathway.The presence of a Segment-A?SA?in the RNase E is a necessary and sufficient factor for the binding of RNase E to the cell membrane.The results of in vivo and in vitro experiments indicate that the destruction of this fragment will cause RNase E to lose its membrane binding ability.PUF family proteins are a class of highly conserved RNA binding proteins,which contain an evolutionarily highly conserved RNA binding domain,which can specifically recognize and bind to specific RNA sequences.Scientists have analyzed the corresponding relationship between the amino acid sequence motif and the recognition nucleotide,laying the foundation for the artificial design of PUF protein and its application in identifying and binding specific RNA sequences.In order to explore whether the degradation rate of mRNA around the inner membrane of E.coli is different from other locations,this study separately designed PUF proteins that bind to the 3'-untranslated regions of ompC and gapA mRNA,and added an inner membrane localization signal to the amino terminus of the PUF protein.The PUF protein binds to the corresponding target gene sequence to locate the inner membrane,and analysis on the correlation between the content of ompC and gapA mRNA and the PUF expression levels was done to determine whether the intracellular location of the E.coli mRNA in the inner membrane affects its degradation rates.The SA is used to localize the PUF protein to the inner membrane of E.coli,and the green fluorescent protein?GFP?is used as a signal to facilitate the location analysis of the PUF.The prokaryotic expression vectors of PUF1-GFP and SA-PUF1-GFP fusion proteins were constructed separately.After the fusion protein expression was induced in E.coli,the distribution of the fusion protein in E.coli was observed with a laser confocal microscope.The result was that SA-PUF1-GFP fusion protein is significantly closer to the inner membrane of E.coli compared with PUF1-GFP.This result indicated that the SA fragment played the function of inner membrane localization and successfully localized the SA-PUF1-GFP fusion protein to the inner membrane of E.coli.Select appropriate E.coli endogenous target m RNA molecules,and verify the interaction between PUF and target RNA molecules in vitro.Using quantitative PCR technology,the mRNA expression levels of the four genes of ompC,gapA,aspC and recA were analyzed,and the relative expression levels of ompC and gapA,which were located in the cell membrane and cytoplasm,re SAectively,were determined to be the mRNA target molecules.Select appropriate sequences in the 3'-untranslated regions?UTR?of ompC and gapA as the PUF recognition sequence,and design and synthesize PUF5?recognizing ompC 3'-untranslated regions 5'-GCGGGCCC-3'?,PUF6?recognizing 3'-untranslated region of gapA 5'-AGAGCGAC-3'?nucleotide sequence.In order to facilitate the subsequent analysis of the interaction between PUF and target oligonucleotides,we designed to use cyan fluorescent protein?CFP?fluorescence and FAM to form a fluorescence energy resonance transfer pair?FRET pair?.We constructed the prokaryotic expression vectors of PUF5-CFP and PUF6-CFP fusion proteins,respectively,and obtained the corresponding fusion proteins through prokaryotic expression and purification.In order to analyze the influence of the distance between the FAM label and the PUF target oligonucleotides on the interaction,we designed and synthesized the 6-FAM modification and the target oligonucleotide with 5,10,5,20 and 25 adenylate away form FAM label(6-FAM-A_5-25-GCGGGCCC/AGAGCGAC).Through the FRET experiment of PUF-CFP and FAM-oligonucleotides,the results show that PUF5 and PUF6 proteins recognize and bind their target RNA sequence,and the affinity is strongest when there is 10 adenylate between the recognition sequence and 6-FAM,K_d values are 0.45 and 0.52?mol/L,respectively.In E.coli cells,the effects of SA-PUF5 and SA-PUF6 expression on the target ompC and gapA mRNA molecular content were analyzed.The prokaryotic expression vectors of PUF5-GFP,SA-PUF5-GFP,PUF6-GFP and SA-PUF6-GFP were constructed,respectively,and the corresponding fusion protein was expressed in E.coli by IPTG induction.The correlations between the expression level of the fusion protein,and the content of target mRNA molecules was further analyzed.It was found that after IPTG induction for 3 hours,PUF5-GFP expression reached 21%of total protein and ompC content decreased to 79.3%of control,while SA-PUF5-GFP expression reached 17%of total protein and ompC content decreased to 61.2%of the control;PUF6-GFP expression reached 19%of the total protein and the gapA content decreased to 84.5%of the control,while the SA-PUF6-GFP expression reached 17%of the total protein and the gapA content decreased to the control's 52.9%.This result indicates that the PUF localized to the inner membrane of E.coli by the SA sequence may bind the target mRNA molecule and relocate it near the inner membrane,and because of the compartment of RNase E in the cell,the degradation rate of target mRNA molecules ompC and gapA is accelerated,and their contents are reduced.Conclusion:Based on the characteristics of membrane localization signal and the specific binding of PUF protein to mRNA molecules,we found that artificially relocalizing mRNA molecules to the inner membrane of E.coli may accelerate its degradation rate and reduce its content.This conclusion provides a new perspective for studying the intracellular degradation of mRNA.