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Depletion of Free 30S Ribosomal Subunits in Escherichia coli by Expression of RNA Containing Shine-Dalgarno-Like Sequences

Mawn, Mary V. and Fournier, Maurille J. and Tirrell, David A. and Mason, Thomas L. (2002) Depletion of Free 30S Ribosomal Subunits in Escherichia coli by Expression of RNA Containing Shine-Dalgarno-Like Sequences. Journal of Bacteriology, 184 (2). pp. 494-502. ISSN 0021-9193. PMCID PMC139575. doi:10.1128/JB.184.2.494-502.2002.

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We have constructed synthetic coding sequences for the expression of poly(α,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.

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Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2002 American Society for Microbiology. Received 31 July 2001; Accepted 16 October 2001. This work was supported by grants from the NSF Polymers and Genetics Programs and by the NSF Materials Research Science and Engineering Center at the University of Massachusetts. We thank Vincent Conticello for early contributions to this work and Paul Bode for his mathematical analysis of sucrose gradient profiles.
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Issue or Number:2
PubMed Central ID:PMC139575
Record Number:CaltechAUTHORS:20111104-135322237
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:27632
Deposited By: Tony Diaz
Deposited On:04 Nov 2011 22:50
Last Modified:09 Nov 2021 16:50

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