Effects of alterations in the translation control region on bacterial gene expression: use of cat gene constructs transcribed from the lac promoter as a model system |
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Authors: | Janet L Schottel John J Sninsky Stanley N Cohen |
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Institution: | Departments of Genetics and Medicine, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. Tel. (415) 497-5315 |
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Abstract: | The region controlling translation of the cat gene, which codes for chloramphenicol acetyltransferase, has been varied structurally in a series of plasmids that place the gene under control of the lac promoter. These plasmid constructs have enabled study of the structural features that affect the efficiency of mRNA translation. Altering the potential for secondary structure formation within the translation control region caused a tenfold variation in the synthesis of CAT enzyme, whereas varying the distance between the Shine-Dalgarno sequence (SD) and the translation start codon from 7 to 13 bases did not significantly affect the yield of CAT. If the SD was situated in a region of mRNA that is capable of base pairing, the efficiency of translation was decreased; however, the translation start codon, AUG, can initiate translation efficiently even when located in a segment capable of duplex formation. Overlapping of the cat translation control region by translation initiated upstream markedly affected initiation of translation within the cat gene: out-of-frame overlapping translation reduced CAT production by 90%; in-frame overlapping translation prevented detectable initiation of protein synthesis at the cat gene translation start codon, and yielded only fusion proteins. The enzymatic activity of such proteins was influenced by the length of the adventitious peptide segment added to the amino-terminus of the CAT polypeptide. |
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Keywords: | Recombinant DNA protein start signals ribosome binding site mRNA plasmid vectors Ap ampicillin bp base pairs CAT chloramphenicol acetyltransferase Cm chloramphenicol DHFR dihydrofolate reductase EtBr ethidium bromide FPV-HA fowl plague virus hemagglutinin kb 1000 bp kcal kilocalories MIC minimal inhibiting concentrations RBS ribosome-binding site SD Shine-Dalgarno sequence SDS sodium dodecyl sulfate |
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