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TRANSLATION EXPRESSION VECTORS
As discussed in Previews Pages, bacterial ribosomes bind mRNA by recognizing the ribosome binding site (RBS) (also known as the Shine-Dalgarno sequence). The RBS base pairs with the sequence AUUCCUCC on the 16S rRNA of the small subunit of the ribosome. The closer the RBS is to the consensus sequence (i.e., UAAGGAGG), the stronger the association. Generally, this leads to more efficient initiation of translation. In addition, for optimal translation, the RBS must be located at the correct distance from the start codon, AUG.
Expression vectors are designed to optimize gene expression at the level of transcription. However, it is also possible to design translational expression vectors to maximize the initiation of translation. These vectors possess a consensus RBS plus an ATG start codon located an optimum distance (8 bp) downstream of the RBS. The cloned gene is inserted into a cloning site that overlaps the start codon. The restriction enzyme NcoI is very convenient because its recognition site (C/CATGG) includes ATG. Therefore it is possible to insert a cloned gene so that its ATG coincides exactly with the ATG of the translational expression vector (Fig. 10.2). The gene to be expressed is cut with NcoI at its 5’-end and with another convenient restriction enzyme at its 3’-end. If necessary, an artificial NcoI site may be introduced into the gene by site-directed mutagenesis or by PCR.
Translational expression vectors also possess a convenient selective marker, usually resistance to ampicillin or some other antibiotic, and a strong, regulated transcription promoter. Downstream of the cloning site are two or three strong terminator sequences, to prevent transcription continuing into plasmid genes.
Although translational expression vectors can optimize the initiation of translation, they do not control the other factors listed earlier, which are properties of the sequence of each individual gene. The secondary structure of the mRNA may have significant effects on the level of translation. If the mRNA folds up so that the RBS and/or start codon are blocked, then translation will be hindered. In particular, the sequence of the first few codons of the coding sequence should be checked for possible base pairing with the region around the RBS. If necessary, bases in the third (redundant) position of each codon may be changed to eliminate such base pairing. Getting active and abundant translation is the key to preventing mRNA instability. Any mRNA that is not being actively translated becomes subject to degradation, which decreases the protein yield.
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