Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Feb 1;29(3):710-5.
doi: 10.1093/nar/29.3.710.

Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable

M O'Connor  1 , W M Lee, A Mankad, C L Squires, A E Dahlberg
Affiliations

Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable

M O'Connor et al. Nucleic Acids Res. .

Abstract

U2449 is one of many invariant residues in the central loop of domain V of 23S rRNA, a region that constitutes part of the peptidyltransferase center of the ribosome. In Escherichia coli, this U is post-transcriptionally modified to dihydrouridine (D) and is the only D modification found in E.coli rRNAs. To analyze the role of this base and its modification in ribosomal function, all three base substitutions were constructed on a plasmid copy of the rrnB operon and assayed for their ability to support cell growth in a strain of E.coli lacking chromosomal rrn operons. Both purine substitution mutations were not viable. However, growth and antibiotic sensitivity of cells expressing only the mutant D2449C rRNA was indistinguishable from wild type. We conclude that while a pyrimidine is required at position 2449 for proper ribosomal function, the D modification is dispensable.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Sequence and secondary structure of the central loop region of domain V of E.coli 23S rRNA. The 10 post-transcriptional modifications are indicated in bold.
Figure 2
Figure 2
Displacement of the resident rrn plasmid, prrnS12, from MC250 by pLK35 (D, wild type at position 2449) and each of the D2449 mutant deriviatives (A, C and G). The MC250 transformants were streaked on LB ampicillin (left) and on LB ampicillin + streptomycin plates (right). Loss of prrnS12 allows expression of rpsL121-associated streptomycin resistance and growth on streptomycin. Streptomycin-resistant colonies were subsequently shown to have lost the prrnS12-associated neomycin resistance.
See this image and copyright information in PMC

References

    1. Maden B.E. (1990) The numerous modified nucleotides in eukaryotic ribosomal RNA. Prog. Nucleic Acid Res. Mol. Biol., 39, 241–303. - PubMed
    1. Ofengand J. and Rudd,K.E. (2000) Bacterial, Archaeal, and organellar rRNA pseudouridines and methylated nucleosides and their enzymes. In Garrett,R.A., Douthwaite,S.R., Liljas,A., Matheson,A.T., Moore,P.B. and Noller,H.F. (eds), The Ribosome: Structure, Function,Antibiotics, and Cellular Interactions. American Society for Microbiology, Washington, DC, pp. 175–189.
    1. Brimacombe R., Mitchell,P., Osswald,M., Stade,K. and Bochkariov,D. (1993) Clustering of modified nucleotides at the functional center of bacterial ribosomal RNA. FASEB J., 7, 161–167. - PubMed
    1. Green R. and Noller,H.F. (1996) In vitro complementation analysis localizes 23S rRNA posttranscriptional modifications that are required for Escherichia coli 50S ribosomal subunit assembly and function. RNA, 2, 1011–1021. - PMC - PubMed
    1. Khaitovich P., Tenson,T., Kloss,P. and Mankin,A.S. (1999) Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA. Biochemistry, 38, 1780–1788. - PubMed

Publication types

LinkOut - more resources