(8.5.5)--SpecializedribosomesystemPrefere建筑设备工程.pdfVIP

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 4762-4766, July 1987 Biochemistry Specialized ribosome system: Preferential translation of a single mRNA species by a subpopulation of mutated ribosomes in Escherichia coli (Shine-Dalgarno sequence/rrnB operon) ANNA HuI AND HERMAN A. DE BOER* Department of Cell Genetics, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080 Communicated by Herbert W. Boyer, March 6, 1987 (receivedfor review November 2, 1986) ABSTRACT InEscherichia coli, all mRNAs are translated In this paper, we describe experiments showing that a by one pool of functionally identical ribosomes. Here, we mutated ribosomal subpopulation can be made and directed describe a system in which a subpopulation of modified to a single mutated mRNA species. This was accomplished ribosomes are directed to a single mutated mRNA species. This by changing the SD sequence on the mRNA as well as the was accomplished by changing the Shine-Dalgarno sequence ASD sequence on the 16S rRNA into entirely different but that precedes the heterologous human growth hormone gene complementary sequences. This system of specialized ribo- from 5 GGAGG to 5 CCTCC or 5 GTGTG. Translation of somes offers apowerful approach to the analysis ofadditional these modified mRNAs by wild-type ribosomes is very ineffi- mutations in crucial regions of the rRNA molecule that cient. When the anti-Shine-Dalgarno region (i.e., the region otherwise would be lethal to the cell. complementary to the Shine-Dalgarno sequence) at the 3 end of the gene encoding 16S rRNA (rrnB) was altered from 5 MATERIALS AND METHODS CCTCC to 5 GGAGG or 5 CACAC, thus restoring its potential to base-pair with the mutated human growth hor- Plasmid amplification was done in E. coli 2