[27] Directing ribosomes to a single mRNA species: A method to study ribosomal RNA mutations and their effects on translation of a single messenger in Escherichia coli

Publisher Summary This chapter discusses directing ribosomes for a single mRNA species and focuses on a method to study ribosomal RNA mutations and their effects on translation of a single messenger in Escherichia Coli . The chapter describes the construction of three plasmids containing the human growth hormone (hGH) gene preceded by an altered Shine–Dalgarno (SD) sequence under the direction of a constitutive trp-promoter. This same plasmid contains an rrnB operon with a mutated anti-Shine–Dalgarno (ASD) sequence (with or without a spectinomycin mutation) under the control of the PL promoter. In each of the three cases (wild type or system VIII, system IX, and system X), the ASD sequence in the rrnB operon is complementary to the SD sequence of the hGH gene of the same plasmid. The chapter describes the several steps related to detailed mutagenesis procedure of double-stranded plasmid DNA. In E. coli , all chromosomal rRNA operons are essentially identical.