Effects of heterologous ribosomal binding sites on the transcription and translation of the lacZ gene of Escherichia coli.
A vector (pKL203) was constructed which contains the promoter-operator region of the lacZ gene and the major part of the coding sequence of the lac operon. The lacZ translation initiation signals [Shine-Dalgarno (SD) sequence and AUG codon] were deleted, and in their place a synthetic linker sequence was inserted, providing single restriction sites for SmaI and BamHI. With this vector constructions were made in which initiation signals of other prokaryotic genes (phage MS2 maturation protein, phage Q beta A2 gene and tufB gene) were fused to the lacZ gene, giving rise to various fusion proteins. The introduction of N-terminal amino acids (aa) in beta-galactosidase (beta-gal) which differ from the wild-type aa invariably leads to an enzyme with a strongly reduced thermostability as compared to the wild-type enzyme. Therefore an immunoprecipitation method was used to measure the amount of fusion protein. It was found that these amounts varied strongly from one construction to another. Concomitant determinations of the amounts of lac-operon-specific mRNA showed an unexpectedly large variation among the clones. No strict correlation could be found between the level of lac mRNA and beta-gal production. Per molecule of lac mRNA, translation appears to be most efficient when the homologous lacZ initiation signal is present.
D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006358:Hot Temperature; D007158:Immunologic Techniques; D007763:Lac Operon; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics* / Q000276:immunology
D012333:RNA, Messenger; D005696:Galactosidases; D001616:beta-Galactosidase