Study Of Lysyl-tRNA Synthetase Promoter

Aminoacyl-tRNA synthetase is a specific enzyme which can catalyze a class of amino acids precursor with tRNA to produce an esterification, Aminoacyl-tRNA synthetase is not only an important class of enzymes in the process of protein synthesis, but also other life activities involved in transcription, translation level regulation,RNA splicing, signal transduction, and immune. There are two kinds of structures completely different lysyl-tRNA synthetase in nature, LysRS-â… , LysRS-â…¡, which responsible for the translation of lysine codons. And Bacillus cereus (Bacillus cereus ATCC14579) in the presence of lysyl-tRNA synthetase I (lysRSI) and lysRSII. lysRSII is the essential gene, but lysRSI is non-essential. LysRSI is not pre-express, but post-expression. LysRSI was regulated strictly by promoter. Therefore, the study of lysRSI promoter is very important to research the biological function of lysRSI genes in B.C.1, We build a different length of lysRSI gene, which knock out promoter 5'end of 300bp, 393bp and 662bp fragment length. Through the genetic engineering methods to connect the GFP after 3'end of promoter and construct gene engineering bacterium which take different length promoter and study the fluorescent we constructed, we found that the GFP did not expressed when knocked out 393bp fragment through experiments, but GFP expression was not affected while 300bp and 662bp fragment was knocked out and the fluorescence intensity enhanced when knocked out 662bp fragments.2, We further studied the lysRSI transcription start site. We find out the lysRSI transcription start site through reverse transcription and increases G in the end, and then sequencing. It located in the 371bp position of 5'end. The transcription start site is in the same of phenomenon we got and explain the observed phenomena. but it did not explain phenomenon of fluorescence when knock out 662bp. So we further investigated the transcription start site of B.cl4579-pUBC-P662-GFP. By the same method, we identified its transcription start site is located on the pUBC19 plasmid.3, We insert the transcription terminator tlt2 into pUBC19 plasmid close to the promoter of the 5'end. After connect different lengths of lysRSI promoter to the transcription terminator tlt2 through genetic engineering approach, we observed the effect on the expression of GFP after inserted the terminator. Through experiments, we can find that the fluorescence intensity of B.cl4579-pUBC-P300-GFP and B.cl4579-pUBC-pLys-GFP was not affected after the terminator inserted, but the fluorescence of B.cl4579-pUBC-P662-GFP is obvious affected. It further prove that the transcription start site is located on the pUBC19 plasmid after knock out 662bp.4, We proceeded six mutations to lysRSI promoter and observed the fluorescent effect in different mutations. The mutations were focus on the stem-loop structure in lysRSI promoter. Through mutation we studied the stem-loop structure effect on the expression of lysRSI gene. T1 mutations have no difference with B.c ptt-pG in LB, but have large differences in the inorganic salt medium. The mutations of T2 and T3 is not fluorescent and T4 mutant present fluorescence in LB, but the fluorescence is weak, T5 and T6 have the same mutation effect, both are part of fluorescence.