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Hybrid Protein between Ribosomal Protein S16 and RimM of Escherichia coli Retains the Ribosome Maturation Function of Both Proteins
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Medicine, Molecular Biology.
2001 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, no 18, 5352-5357 p.Article in journal (Refereed) Published
Abstract [en]

The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes and is important for efficient maturation of the 30S subunits. A mutant lacking RimM shows a sevenfold-reduced growth rate and a reduced translational efficiency. Here we show that a double alanine-for-tyrosine substitution in RimM prevents it from associating with the 30S subunits and reduces the growth rate of E. coli approximately threefold. Several faster-growing derivatives of the rimM amino acid substitution mutant were found that contain suppressor mutations which increased the amount of the RimM protein by two different mechanisms. Most of the suppressor mutations destabilized a secondary structure in the rimM mRNA, which previously was shown to decrease the synthesis of RimM by preventing the access of the ribosomes to the translation initiation region on the rimM mRNA. Three other independently isolated suppressor mutations created a fusion between rpsP, encoding the ribosomal protein S16, and rimM on the chromosome as a result of mutations in the rpsP stop codon preceding rimM. A severalfold-higher amount of the produced hybrid S16-RimM protein in the suppressor strains than of the native-sized RimM in the original substitution mutant seems to explain the suppression. The S16-RimM protein but not any native-size ribosomal protein S16 was found both in free 30S ribosomal subunits and in translationally active 70S ribosomes of the suppressor strains. This suggests that the hybrid protein can substitute for S16, which is an essential protein probably because of its role in ribosome assembly. Thus, the S16-RimM hybrid protein seems capable of carrying out the important functions that native S16 and RimM have in ribosome biogenesis.

Place, publisher, year, edition, pages
2001. Vol. 183, no 18, 5352-5357 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:umu:diva-4314DOI: 10.1128/JB.183.18.5352-5357.2001PubMedID: 11514519OAI: diva2:143344
Available from: 2004-12-15 Created: 2004-12-15 Last updated: 2010-08-06Bibliographically approved
In thesis
1. Accessory factors for ribosomal assembly
Open this publication in new window or tab >>Accessory factors for ribosomal assembly
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The assembly of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins) into ribosomal subunits (30S and 50S) is a complex process. Transcription of rRNA requires antitermination proteins and the primary transcripts are processed by ribonucleases. R-proteins and rRNAs are chemically modified, the r-proteins bind to the rRNAs and the formed RNA-protein complexes are folded into mature ribosomal subunits. All these processes are well-coordinated and overlapping. Non-ribosomal factors are required for proper assembly and maturation of the ribosomal subunits. Two of these factors are the RimM and RbfA proteins, which bind to 30S subunits and are important for efficient processing of 16S rRNA. Lack of either RimM or RbfA results in a reduced amount of polysomes and a lower growth rate. An increased amount of RbfA can partially compensate for deficiencies shown by a RimM lacking mutant.

Here, mutations that alter phylogenetically conserved amino acids in RimM have been constructed. One of these (rimM120), which resulted in the replacement of two adjacent tyrosines by alanines, reduced the growth rate three-fold and also decreased the processing efficiency of 16S rRNA. The RimM120 mutant protein showed a much reduced binding to the 30S subunits. Suppression of the rimM120 mutant was achieved by increased amount of the RimM120 protein, by overexpression of rbfA, or by mutations that changed r-protein S19 or 16S rRNA. A variant of r-protein S13, which was previously isolated as a suppressor to a deletion of rimM (∆rimM), suppressed also the rimM120 mutation. The wild-type RimM protein, but not the RimM120 protein, was shown to bind r-protein S19 in the 30S subunits. The changes in S13, S19 and 16S rRNA that compensated for the deficiencies shown by the rimM mutants are all located within a small region of the head of the 30S subunit, suggesting that this region is the likely target for the RimM action.

To isolate RbfA variants that show reduced association with the 30S subunits, phylogenetically conserved, surface exposed amino acid residues of RbfA were changed to alanines or, in some instances, to amino acids of the opposite charge to that in the wild-type protein. Alterations of F5, R31, D46 and D100 had the largest effect on growth.

Mutations in the metY-nusA-infB operon, isolated as suppressors to the ∆rimM mutant, were shown to increase the amounts of RbfA. In a ∆rimM mutant, all RbfA protein was found associated with the 30S subunits and no free RbfA was detected.

The RlmB protein was shown to be the methyltransferase responsible for the formation of Gm2251 in 23S rRNA in Escherichia coli. Unlike a Saccharomyces cerevisiae mutant that lacks the orthologue to RlmB, Pet56p, which methylates mitochondrial rRNA, a ∆rlmB mutant did not show any defects in ribosomal assembly.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2004. 57 p.
Molecular biology, RimM, RbfA, RlmB, ribosomal assembly, rRNA processing, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:umu:diva-385 (URN)91-7305-783-5 (ISBN)
Public defence
2005-01-14, major groove, 6L, NUS, Institutionen för molekylärbiologi, 901 87 Umeå, Umeå, 13:00
Available from: 2004-12-15 Created: 2004-12-15 Last updated: 2010-08-06Bibliographically approved

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Lövgren, J. MattiasWikström, P. Mikael
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