Selection for intragenic suppressors of lethal 23S rRNA mutations in Escherichia coli identifies residues important for ribosome assembly and function |
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Authors: | Michael O’Connor |
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Institution: | (1) School of Biological Sciences, University of Missouri-Kansas City, 5007 Rockhill Rd, Kansas City, MO 64110, USA |
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Abstract: | Mutations in several functionally important regions of the 23S rRNA of E. coli increase the levels of frameshifting and readthrough of stop codons. These mutations include U2555A, U2555G, ΔA1916 and U2493C.
The mutant rRNAs are lethal when expressed at high levels from a plasmid, in strains also expressing wild type rRNA from chromosomal
rrn operons. The lethal phenotype can be suppressed by a range of second-site mutations in 23S rRNA. However, analysis of the
functionality of the double mutant rRNAs in heterogeneous ribosome populations shows that in general, the second site mutations
do not restore function. Instead, they prevent the assembly, or entry of the mutant 50S subunits into the functioning 70S
ribosome and polysome pools, by affecting the competitiveness of the mutant subunits for association with 30S particles. The
second-site mutations lie in regions of the 23S rRNA involved in subunit assembly, intersubunit bridge formation and interactions
of the ribosome with tRNAs and factors. These second site suppressor mutations thus define functionally important rRNA nucleotides
and this approach may be of general use in the functional mapping of large RNAs. |
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Keywords: | Ribosome Inter-subunit bridge Suppressor rRNA lethal mutation Decoding fidelity |
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