Depletion of Free 30S Ribosomal Subunits in Escherichia coli by Expression of RNA Containing Shine-Dalgarno-Like Sequences
Abstract
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.
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.Attached Files
Published - MAWjbact02.pdf
Files
Name | Size | Download all |
---|---|---|
md5:966d5fb2a75733b58a32841af65513bc
|
957.0 kB | Preview Download |
Additional details
- PMCID
- PMC139575
- Eprint ID
- 27632
- Resolver ID
- CaltechAUTHORS:20111104-135322237
- NSF
- Created
-
2011-11-04Created from EPrint's datestamp field
- Updated
-
2021-11-09Created from EPrint's last_modified field