Depletion of free 30S ribosomal subunits in Escherichia coli by expression of RNA containing Shine-Dalgarno-like sequences.
We have constructed synthetic coding sequences for the expression of poly(alpha,L-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5'-GAGGAGG-3') that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA.
Mawn
Mary V
MV
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Fournier
Maurille J
MJ
Tirrell
David A
DA
Mason
Thomas L
TL
D001483:Base Sequence; D002455:Cell Division; D002499:Centrifugation, Density Gradient; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D011099:Polyglutamic Acid / Q000235:genetics*; D014176:Protein Biosynthesis; D011691:Puromycin / Q000494:pharmacology; D012329:RNA, Bacterial / Q000502:physiology*; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*; D013395:Sucrose; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D020506:Untranslated Regions
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D011993:Recombinant Fusion Proteins; D020506:Untranslated Regions; D011099:Polyglutamic Acid; D007544:Isopropyl Thiogalactoside; D011691:Puromycin; D013395:Sucrose; D013762:Tetrahydrofolate Dehydrogenase