Skip Navigation

This Article
Right arrow Print PDF (1270K)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Poot, R. A.
Right arrow Articles by Duin, J. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poot, R. A.
Right arrow Articles by Duin, J. V.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 1993, Vol. 21, No. 23 5398-5402
© 1993

MOLECULAR BIOLOGY

Separation of mutant and wild-type ribosomes based on differences in their anti Shine - Dalgarno sequence

Raymond A. Poot, Marcel F. Brink, Cornelis W.A. Pleji, Herman A.de Boer and Jan Van Duin*

Department of Biochemistry, Gorlaeus Laboratories, University of Leiden PO Box 9502, 2300 RA Leiden, The Netherlands

*To whom correspondence should be addressed

Received August 19, 1993. Revised October 19, 1993. Accepted October 19, 1993.

We describe a system to isolate 30S ribosomal subunits which contain targeted mutations in their 16S rRNA. The mutations of interest should be present in so-called specialized 30S subunits which have an anti- Shine - Dalgarno sequence that is altered from 5' ACCUCC to 5' ACACAC. These plasmid-encoded specialized 30S subunits are separated from their chromosomally encoded wild-type counterparts by affinity chromatography that exploits the different Shine - Dalgarno complementarity. An oligonucleotide complementary to the 3' end of wild-type 16S rRNA and attached to a solid phase matrix retains the wild-type 30S subunits. The flow-through of the column contains close to 100% mutant 30S subunits. Toeprinting assays demonstrate that affinity column treatment does not cause significant loss of activity of the specialized particles in initiation complex formation, whereas elongation capacity as determined by poly(Phe) synthesis is only slightly decreased. The method described offers an advantage over total reconstitution from in vitro transcribed mutant 16S rRNA since our 30S subunits contain the naturally occurring base modifications in their 16S rRNA.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
R. A. Poot, N. V. Tsareva, I. V. Boni, and J. van Duin
RNA folding kinetics regulates translation of phage MS2 maturation gene
PNAS, September 16, 1997; 94(19): 10110 - 10115.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.