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Accessory factors for ribosomal assembly
Umeå University, Faculty of Medicine, Molecular Biology.
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.
Keyword [en]
Molecular biology, RimM, RbfA, RlmB, ribosomal assembly, rRNA processing
Keyword [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-385ISBN: 91-7305-783-5 (print)OAI: oai:DiVA.org:umu-385DiVA: diva2:143348
Public defence
2005-01-14, major groove, 6L, NUS, Institutionen för molekylärbiologi, 901 87 Umeå, Umeå, 13:00
Opponent
Supervisors
Available from: 2004-12-15 Created: 2004-12-15 Last updated: 2010-08-06Bibliographically approved
List of papers
1. Hybrid Protein between Ribosomal Protein S16 and RimM of Escherichia coli Retains the Ribosome Maturation Function of Both Proteins
Open this publication in new window or tab >>Hybrid Protein between Ribosomal Protein S16 and RimM of Escherichia coli Retains the Ribosome Maturation Function of Both Proteins
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4314 (URN)10.1128/JB.183.18.5352-5357.2001 (DOI)11514519 (PubMedID)
Available from: 2004-12-15 Created: 2004-12-15 Last updated: 2010-08-06Bibliographically approved
2. Characterization of mutations in the metY-nusA-infB operon that suppress the slow growth of a DeltarimM mutant.
Open this publication in new window or tab >>Characterization of mutations in the metY-nusA-infB operon that suppress the slow growth of a DeltarimM mutant.
2001 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, no 20, 6095-6106 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. A DeltarimM mutant shows a sevenfold-reduced growth rate and a reduced translational efficiency, probably as a result of aberrant assembly of the ribosomal 30S subunits. The slow growth and translational deficiency can be partially suppressed by increased synthesis of the ribosome binding factor RbfA. Here, we have identified 14 chromosomal suppressor mutations that increase the growth rate of a DeltarimM mutant by increasing the expression of rbfA. Nine of these mutations were in the nusA gene, which is located upstream from rbfA in the metY-nusA-infB operon; three mutations deleted the transcriptional terminator between infB and rbfA; one was an insertion of IS2 in infB, creating a new promoter for rbfA; and one was a duplication, placing a second copy of rbfA downstream from a promoter for the yhbM gene. Two of the nusA mutations were identical, while another mutation (nusA98) was identical to a previously isolated mutation, nusA11, shown to decrease termination of transcription. The different nusA mutations were found to increase the expression of rbfA by increasing the read-through of two internal transcriptional terminators located just downstream from the metY gene and that of the internal terminator preceding rbfA. Induced expression of the nusA(+) gene from a plasmid in a nusA(+) strain decreased the read-through of the two terminators just downstream from metY, demonstrating that one target for a previously proposed NusA-mediated feedback regulation of the metY-nusA-infB operon expression is these terminators. All of the nusA mutations produced temperature-sensitive phenotypes of rimM(+) strains. The nusA gene has previously been shown to be essential at 42 degrees C and below 32 degrees C. Here, we show that nusA is also essential at 37 degrees C.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4315 (URN)10.1128/JB.183.20.6095-6106.2001 (DOI)11567010 (PubMedID)
Available from: 2004-12-15 Created: 2004-12-15 Last updated: 2010-08-06Bibliographically approved
3. The rlmB Gene Is Essential for Formation of Gm2251 in 23S rRNA but Not for Ribosome Maturation in Escherichia coli
Open this publication in new window or tab >>The rlmB Gene Is Essential for Formation of Gm2251 in 23S rRNA but Not for Ribosome Maturation in Escherichia coli
2001 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, no 23, 6957-6960 p.Article in journal (Refereed) Published
Abstract [en]

In Saccharomyces cerevisiae, the rRNA Gm2270 methyltransferase, Pet56p, has an essential role in the maturation of the mitochondrial large ribosomal subunit that is independent of its methyltransferase activity. Here we show that the proposed Escherichia coli ortholog, RlmB (formerly YjfH), indeed is essential for the formation of Gm in position 2251 of 23S rRNA. However, a DeltarlmB mutant did not show any ribosome assembly defects and was not outgrown by a wild-type strain even after 120 cell mass doublings. Thus, RlmB has no important role in ribosome assembly or function in E. coli.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4316 (URN)10.1128/JB.183.23.6957-6960.2001 (DOI)11698387 (PubMedID)
Available from: 2004-12-15 Created: 2004-12-15 Last updated: 2010-08-06Bibliographically approved
4. The PRC-barrel domain of the ribosome maturation protein RimM mediates binding to ribosomal protein S19 in the 30S ribosomal subunits.
Open this publication in new window or tab >>The PRC-barrel domain of the ribosome maturation protein RimM mediates binding to ribosomal protein S19 in the 30S ribosomal subunits.
Show others...
2004 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 10, no 11, 1798-1812 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. A DeltarimM mutant is defective in 30S maturation and accumulates 17S rRNA. To study the interaction of RimM with the 30S and its involvement in 30S maturation, RimM amino acid substitution mutants were constructed. A mutant RimM (RimM-YY-->AA), containing alanine substitutions for two adjacent tyrosines within the PRC beta-barrel domain, showed a reduced binding to 30S and an accumulation of 17S rRNA compared to wild-type RimM. The (RimM-YY-->AA) and DeltarimM mutants had significantly lower amounts of polysomes and also reduced levels of 30S relative to 50S compared to a wild-type strain. A mutation in rpsS, which encodes r-protein S19, suppressed the polysome- and 16S rRNA processing deficiencies of the RimM-YY-->AA but not that of the DeltarimM mutant. A mutation in rpsM, which encodes r-protein S13, suppressed the polysome deficiency of both rimM mutants. Suppressor mutations, found in either helices 31 or 33b of 16S rRNA, improved growth of both the RimM-YY-->AA and DeltarimM mutants. However, they suppressed the 16S rRNA processing deficiency of the RimM-YY-->AA mutant more efficiently than that of the DeltarimM mutant. Helices 31 and 33b are known to interact with S13 and S19, respectively, and S13 is known to interact with S19. A GST-RimM but not a GST-RimM(YY-->AA) protein bound strongly to S19 in 30S. Thus, RimM likely facilitates maturation of the region of the head of 30S that contains S13 and S19 as well as helices 31 and 33b.

Keyword
Alanine/metabolism, Amino Acid Sequence, Amino Acid Substitution, Bacterial Proteins/*chemistry/genetics/*metabolism, Escherichia coli/genetics/growth & development, Escherichia coli Proteins/*chemistry/genetics/*metabolism, Gene Expression Regulation; Bacterial, Glutathione Transferase/metabolism, Models; Molecular, Molecular Sequence Data, Mutagenesis; Site-Directed, Mutation, Protein Structure; Tertiary, RNA Processing; Post-Transcriptional, RNA; Ribosomal; 16S/genetics/metabolism, RNA-Binding Proteins, Recombinant Proteins/metabolism, Ribosomal Proteins/*chemistry/genetics/*metabolism, Ribosomes/*metabolism, Sequence Homology; Amino Acid, Tyrosine/metabolism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-16783 (URN)10.1261/rna.7720204 (DOI)15496525 (PubMedID)
Available from: 2007-10-11 Created: 2007-10-11 Last updated: 2010-08-06Bibliographically approved

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