Only one out of the three strong ribosomal binding sites of the early region of bacteriophage T7 exhibits high translational efficiency in fragments of about 30 base pairs.
Within the early region of bacteriophage T7 three genes, 0.3, 1 and 1.3, are most efficiently expressed. They belong to the strongest initiation signals of Escherichia coli. In the T7 wild-type situation the proteins are produced with a molar ratio of gene 1:1.3:0.3 protein = 1:3.9:9.7. DNA fragments of about 30 base pairs comprising the ribosomal binding sites (RBS) of these genes were synthesized and cloned into derivatives of the pDS1 vector ribosomal binding sites (RBS) of these genes were synthesized and cloned into two derivatives of the pDS1 vector just upstream of the mouse dihydrofolate reductase gene. Although all tested RBS fragments contained an initiation triplet, a Shine-Dalgarno sequence and some nucleotides upstream and downstream of this region, only the gene 1.3 RBS fragment showed high efficiency whereas those of genes 0.3 and 1 were at the border of significance. The amount of synthesized mRNA was about the same for all three constructs. A major influence of vector-derived sequences on the RBS activity could be ruled out. The high translational activity of the short 1.3 gene RBS seems to be largely due to its primary structure. The other two RBSs studied require much longer sequences for high activity.
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005796:Genes; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010446:Peptide Fragments / Q000032:analysis; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D012269:Ribosomal Proteins / Q000032:analysis*; D012270:Ribosomes / Q000032:analysis*; D013604:T-Phages / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D010446:Peptide Fragments; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D013762:Tetrahydrofolate Dehydrogenase