The Effect On Translation Efficiency Of MRNA Secondary Structure

Translation is one of the key processes of gene expression and its efficiency is very important for life. As a crucial part of translation system, mRNA has the effect on the rate of translation while encoding protein. mRNA secondary structure could regulate the translation speed. Understanding the effect of mRNA on the rate of translation helps to reveal the regulation mechanism of translation efficiency, to deepen the comprehension of the evolution and to improve the transgenic protein production in the field of biotechnology.The sequence of APA1 gene was cloned from S288 c by PCR, connected with EGFP gene, and inserted into pY16 vector. The vector was used for synonymous mutations by One-step site-directed mutagenesis, and mutations which can change mRNA secondary structure of APA1 gene were obtained.Testing the fluorescence intensity of YS59 transformed by vector containing mutations elucidated the relationship between mRNA secondary structure and translation speed. The main research results are as follows:1. The fragment of APA1 and EGFP were digested with restriction enzyme and connected with digested pY16 vector. Enzyme digestion and sequencing results showed that the expression vector pY16TEF1-EGFP-APA1 was successfully constructed. Testing the fluorescence intensity of YS59 transformed by vector pY16TEF1-EGFP-APA1 with fluorescence microscope proved that APA1 with EGFP gene were co-expressed.2. 18 synonymous mutations which can change mRNA secondary structure of APA1 were introduced into expression vector pY16TEF1-EGFP-APA1 by One-step site-directed mutagenesis.Compared with sequence existed into database, sequencing results showed mutations were successfully introduced. One-step site-directed mutagenesis is an effective method. Testing the fluorescence intensity of YS59 transformed into vector containing mutations with fluorescence microscope indicated that these APA1 genes containing mutations could be expressed.3. The fluorescence intensity of YS59 transformed by synonymous mutationvector was detected by flow cytometry. The results elucidated that mRNA secondary structure could influence the protein translation and even translation elongation whether the prediction structure was near translation starting area or close to the end zone. To some extent, the damage of mRNA secondary structure could enhance translation efficiency. It was surprised that not all the cases showed the same results.The mutation which did not change fluorescence intensity heavily even reduced the fluorescence intensity illustrated that the effects of secondary structure on translation rate were complicated and still had a far way to go. Meanwhile, the fact that the mutation which did not change mRNA secondary structure changing the fluorescence intensity told that there were some unknown factors which had effect on translation speed.