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Removal and Replacement of Ribosomal Proteins: Effects on Bacterial Fitness and Ribosome Function
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Dan Andersson)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein synthesis is a complex process performed by sophisticated cellular particles known as ribosomes. Although RNA constitutes the major structural and functional component, ribosomes from all kingdoms contain an extensive array of proteins with largely undefined functional roles. The work presented in this thesis addresses ribosomal complexity using mutants of Salmonella typhimurium to examine the physiological effects of ribosomal protein (r-protein) removal and orthologous replacement on bacterial fitness and ribosome function.

The results of paper I demonstrate that removal of small subunit protein S20 conferred two independent translation initiation defects: (i) a significant reduction in the rate and extent of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in subunit association. The topographical location of S20 in mature 30S subunits suggests that these perturbations are the result of improper orientation of helix 44 of the 16S rRNA when S20 is absent. In paper II we show that the major functional impairment associated with loss of large subunit protein L1 manifested as an increase in free ribosomal subunits at the expense of translationally active 70S particles. Furthermore, the formation of free ribosomal subunits was imbalanced suggesting that L1 is required to suppress degradation or promote formation of 30S subunits. Compensatory evolution revealed that mutations in other large subunit proteins mitigate the cost of L1 removal, in one case seemingly via an increase in 70S complex formation. As shown in paper III, the large fitness costs associated with complete removal of r-proteins is in contrast to the generally mild costs of orthologous protein replacement, even in the absence of a high degree of homology to the native protein. This clearly demonstrates the robustness and plasticity of the ribosome and protein synthesis in general and it also implies that functional constraints are highly conserved between these proteins. The findings of paper III also allowed us to examine the barriers that constrain horizontal gene transfer and we find that increased gene dosage of the sub-optimal heterologous protein may be an initial response to stabilize deleterious transfer events. Overall the results highlight the requirement of r-proteins for the maintenance of ribosomal structural integrity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 93 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 667
Keyword [en]
ribosome, protein synthesis, ribosomal proteins, translation initiation, ribosome biogenesis, fitness costs, compensatory evolution, horizontal gene transfer
National Category
Microbiology in the medical area Biochemistry and Molecular Biology
Research subject
URN: urn:nbn:se:uu:diva-150401ISBN: 978-91-554-8061-5 (print)OAI: diva2:408601
Public defence
2011-05-20, C8:301, BMC, Husargatan 3, Uppsala, 09:00 (English)
Available from: 2011-04-28 Created: 2011-03-29 Last updated: 2011-05-05
List of papers
1. Ribosomes lacking protein S20 are defective in mRNA binding and subunit association
Open this publication in new window or tab >>Ribosomes lacking protein S20 are defective in mRNA binding and subunit association
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2010 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, no 3, 767-776 p.Article in journal (Refereed) Published
Abstract [en]

The functional significance of ribosomal proteins is still relatively unclear. Here, we examined the role of small subunit protein S20 in translation using both in vivo and in vitro techniques. By means of lambda red recombineering, the rpsT gene, encoding S20, was removed from the chromosome of Salmonella enterica var. Typhimurium LT2 to produce a DeltaS20 strain that grew markedly slower than the wild type while maintaining a wild-type rate of peptide elongation. Removal of S20 conferred a significant reduction in growth rate that was eliminated upon expression of the rpsT gene on a high-copy-number plasmid. The in vitro phenotype of mutant ribosomes was investigated using a translation system composed of highly active, purified components from Escherichia coli. Deletion of S20 conferred two types of initiation defects to the 30S subunit: (i) a significant reduction in the rate of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in association with the 50S subunit. Both of these impairments were partially relieved by an extended incubation time with mRNA, fMet-tRNA(fMet), and initiation factors, indicating that absence of S20 disturbs the structural integrity of 30S subunits. Considering the topographical location of S20 in complete 30S subunits, the molecular mechanism by which it affects mRNA binding and subunit docking is not entirely obvious. We speculate that its interaction with helix 44 of the 16S ribosomal RNA is crucial for optimal ribosome function.

National Category
Medical and Health Sciences Biological Sciences
urn:nbn:se:uu:diva-135217 (URN)10.1016/j.jmb.2010.02.004 (DOI)000276177300012 ()20149799 (PubMedID)
Available from: 2010-12-06 Created: 2010-12-06 Last updated: 2017-12-12Bibliographically approved
2. Ribosomal protein L1 is required for balanced formation of ribosomal subunits
Open this publication in new window or tab >>Ribosomal protein L1 is required for balanced formation of ribosomal subunits
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(English)Manuscript (preprint) (Other academic)
ribosomal protein L1, ribosome biogenesis, compensatory evolution
National Category
Cell and Molecular Biology Microbiology in the medical area Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
urn:nbn:se:uu:diva-150400 (URN)
Available from: 2011-03-29 Created: 2011-03-29 Last updated: 2016-11-30
3. Compensatory gene amplification restores fitness after inter-species gene replacements
Open this publication in new window or tab >>Compensatory gene amplification restores fitness after inter-species gene replacements
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2010 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 75, no 5, 1078-1089 p.Article in journal (Refereed) Published
Abstract [en]

Genes introduced by gene replacements and other types of horizontal gene transfer (HGT) represent a significant presence in many archaeal and eubacterial genomes. Most alien genes are likely to be neutral or deleterious upon arrival and their long-term persistence may require a mechanism that improves their selective contribution. To examine the fate of inter-species gene replacements, we exchanged three native S. typhimurium genes encoding ribosomal proteins with orthologues from various other microbes. The results show that replacement of each of these three genes reduces fitness to such an extent that it would provide an effective barrier against inter-species gene replacements in eubacterial populations. However, these fitness defects could be partially ameliorated by gene amplification that augmented the dosage of the heterologous proteins. This suggests that suboptimal expression is a common fitness constraint for inter-species gene replacements, with fitness costs conferred by either a lower expression level of the alien protein compared with the native protein or a requirement for an increased amount of the alien protein to maintain proper function. Our findings can explain the observation that duplicated genes are over-represented among horizontally transferred genes, and suggest a potential coupling between compensatory gene amplification after HGT and the evolution of new genes.

National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-131545 (URN)10.1111/j.1365-2958.2009.07030.x (DOI)000274808800004 ()20088865 (PubMedID)
Available from: 2010-10-05 Created: 2010-10-05 Last updated: 2017-12-12Bibliographically approved

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