Separation of Mutant and Wild-type Ribosomes Based on Differences in Their Anti Shine-Dalgarno Sequence

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1993 Nov 25

PMID 8265355

Citations 4

Authors

R A Poot

M F Brink

C W Pleij

H A DE BOER

J van Duin

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Base complementarity in helix 2 of the central pseudoknot in 16S rRNA is essential for ribosome functioning.

Poot R, van den Worm S, Pleij C, van Duin J Nucleic Acids Res. 1998; 26(2):549-53.

PMID: 9421514 PMC: 147307. DOI: 10.1093/nar/26.2.549.

RNA folding kinetics regulates translation of phage MS2 maturation gene.

Poot R, Tsareva N, Boni I, van Duin J Proc Natl Acad Sci U S A. 1997; 94(19):10110-5.

PMID: 9294171 PMC: 23320. DOI: 10.1073/pnas.94.19.10110.

The central pseudoknot in 16S ribosomal RNA is needed for ribosome stability but is not essential for 30S initiation complex formation.

Poot R, Pleij C, van Duin J Nucleic Acids Res. 1996; 24(19):3670-6.

PMID: 8871543 PMC: 146175. DOI: 10.1093/nar/24.19.3670.

A translational fidelity mutation in the universally conserved sarcin/ricin domain of 25S yeast ribosomal RNA.

Liu R, Liebman S RNA. 1996; 2(3):254-63.

PMID: 8608449 PMC: 1369368.

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