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Journal of Bacteriology, January 2002, p. 494-502, Vol. 184, No. 2
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.2.494-502.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Mary V. Mawn,1 Maurille J. Fournier,1 David A. Tirrell,2,{dagger} and Thomas L. Mason1

Department of Biochemistry and Molecular Biology,1 Department of Polymer Science and Engineering, University of Massachusetts, Amherst, Massachusetts 010032

Received 31 July 2001/ Accepted 16 October 2001

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.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Lederle Graduate Research Center, University of Massachusetts, Box 34505, Amherst, MA 01003-4505. Phone: (413) 545-3122. Fax: (413) 545-3291. E-mail: tmason{at}biochem.umass.edu.

{dagger} Present address: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.

Journal of Bacteriology, January 2002, p. 494-502, Vol. 184, No. 2
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.2.494-502.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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