Excavation Of Saccharomyces Cerevisiae Translational Regulation Elements And Functional Characterization

Protein translation process is one of the most fundamental processes of life, and the translation level is largely decisived by translation initiation. Recent studies have found that the 5’ untranslated area(5’UTR) of mRNA can significantly influence protein translation initiation, thus adjustment protein expression levels. It is well accepted that 5’UTR includes important protein translation regulatory elements, yet the regulatory role of 5’UTR on protein translation haven’t been studied clearly. In this study, a high-throughput 5’UTR expression and characterization system in Saccharomyces cerevisiae was constructed, and random synthetic 5’UTR sequence library based on random primers mix was expressed and characterized through this system. After that, the protein expression data collected from FACS was mapped to 5’UTR sequence data collected form high-throughput sequencing, and then the regulatory role of each 5’UTR sequence on translation initiation was messured and analysised to indentify sequence elements or structure elements that play a specific regulatory role in translation process. All these work were aiming to clarify the regulatory mechanism of 5’UTR on protein translation initiation in Saccharomyces cerevisiae. The main works were listed following:(1) Construction 5’UTR sequence expression element with molecular cloning. This study used two different methods to acquire the Saccharomyces cerevisiae cells co-expressing red fluorescent protein mCherry and green fluorescent protein GFP. In the first expression style, red fluorescent protein gene mCherry was integrated into the host cell genome first, and then the green fluorescent protein gene GFP was transformed and integrated into recombinant host strain; the other co-expression style was conducted by expression GFP and mCherry genes in one shuttle plasmid, and transformed to a wild type yeast strain. Analysis of fluorescent protein expression,the results shows that geneome expression result in intence and steady expression, while plasmid expression may lead to a wide range of fluorescence,more suitable for library construction.(2) Five 50-bp length UTR genes selection. UTR genes with differences expression efficiency were inserted into vector YIplac211 –RPL8A-GFP, and then those 5’UTR sequences which can significantly affect the expression of green fluorescent protein were selected.(3) The use of error-prone PCR method to constructed the 5’UTR sequence- protein expression mutants library.(4) YLR167 W gene mutants library construction and selection. By random synthesis of 5 length of 89 bp ssDNA、containing 12 random nucleotides and restriction sites, the protect base and the start codon of single-stranded DNA. The single-stranded DNA as a template, PCR amplification of the random 5’UTR sequence to form a 5’UTR random sequence DNA library.The random sequence DNA and expression vector were together transformed to a wild type yeast strain.Sorting 5’UTR library by FACS technology,and divided into 10 groups based on green fluorescent protein intensity. FACS analysis of different groups of cells showed a 40-fold in difference protein expression.