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The RimP protein is important for maturation of the 30S ribosomal subunit
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Wikström)
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Wikström)
2009 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 386, no 3, 742-753 p.Article in journal (Refereed) Published
Abstract [en]

The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (DeltarimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 degrees C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.

Place, publisher, year, edition, pages
2009. Vol. 386, no 3, 742-753 p.
Keyword [en]
RimP, YhbC, 30S maturation, ribosome assembly, 16S rRNA processing
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:umu:diva-33104DOI: 10.1016/j.jmb.2008.12.076PubMedID: 19150615OAI: oai:DiVA.org:umu-33104DiVA: diva2:310101
Available from: 2010-04-12 Created: 2010-04-12 Last updated: 2017-12-12Bibliographically approved
In thesis
1. The importance of maturation factors in 30S ribosomal subunit assembly
Open this publication in new window or tab >>The importance of maturation factors in 30S ribosomal subunit assembly
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The assembly of the ribosome is a complex process that needs to be highly efficient to support maximum growth. Although the individual subunits of the ribosome can be reconstituted in vitro, such a reaction is inefficient in comparison to the assembly rate in vivo. What differentiates the in vivo from the in vitro assembly is primarily the presence of ribosome assembly proteins. These are proteins that assist in the assembly of the ribosomal subunits but are not part of the mature ribosome. In bacteria, the ribosome assembly proteins include rRNA processing enzymes and rRNA/ribosomal protein (r-protein) modifying enzymes. One set of ribosome assembly proteins, the ribosome maturation factors, have been difficult to classify due to their differences in structure and their apparent lack of similarities with regard to function. As part of this thesis, the previously uncharacterized RimP (ribosome maturation) protein formerly known as P15A or YhbC, was studied. Deletion of the rimP gene affected the growth rate more severely at 44°C than at 37°C and 30°C. Polysome profile analysis revealed a decrease in the amount of translating ribosomes and a corresponding increase in the amount of free 50S and 30S ribosomal subunits. The disproportionate large increase in 50S relative to 30S subunits indicated a 30S assembly defect. RimP was shown to localize to the 30S ribosomal subunit, and an accumulation of 17S rRNA, a precursor to 16S rRNA, supports a role for RimP in 30S subunit maturation. The results from in vitro reconstitution experiments have given valuable insights in the assembly of the 30S subunit. By using a recently developed method, the role of ribosome maturation factors Era, RimM and RimP during in vitro reconstitutions of the 30S subunit was investigated. Era was found to increase the incorporation rate for most of the late binding r-proteins, while RimM and RimP had more specific effects. RimM increased the incorporation rate for r-proteins S19 and S9 and inhibited the incorporation of S13 and S12, whereas RimP increased the incorporation rate primarily for S12 and S5. A comparison of the ribosome maturation factors RimP and RbfA (ribosome binding factor A) revealed structural similarities between the N-terminal domain of RimP and the single domain of RbfA. RbfA is a 15 kDa protein that was found to high copy-suppress a dominant C23U 16S rRNA mutation giving rise to cold-sensitivity in E. coli. A number of chromosomal suppressor mutations that increased the growth rate of an rbfA null mutant were isolated. The five strongest suppressor mutations were localized to the rpsE gene, for r-protein S5 and resulted in amino acid substitutions in three positions: G87A, G87S, G91A, A127V and A127T. These alterations improved translation and the processing of 16S rRNA in the rbfA null mutant. Moreover, they also suppressed the slow growth of the C23U rRNA mutant at 30, 37 and 44°C.

Abstract [sv]

Monteringen av ribosomen är en komplex process som måste vara effektiv för cellen skall kunna växa så fort som möjligt. Det är visat sedan tidigare att ribosomens två subenheter kan monteras ihop in vitro och sedan vara del av en ribosom som gungerar vid proteinsyntes, dock är den typen av rekonstrueringsreaktioner mycket ineffektiva i jämförelse med vad som krävs in vivo. Skillnaden mellan dessa två tillstånd är primärt in vivo-reaktionens närvaro av hjälpproteiner. Hjälpproteinerna assisterar monteringen av ribosomens subenheter men är själva inte en del av den färdiga ribosomen. Inom denna klass av proteiner återfinns proteiner som t.ex. processerar ribosomalt RNA och proteiner som modifierar ribosomalt RNA och ribosomala protein. En klass av hjälpproteiner, mognadsfaktorerna, har varit svåra att klassificera på grund av strukturella olikheter och en brist på funktionella likheter. En del i detta avhandlingsarbete var karaktäriseringen av den tidigare okända mognadsfaktorn RimP, tidigare kallad YhbC eller P15A. En deletion av rimP hade störst påverkan på tillväxthastigheten vid 44°C, men effekter kunde även ses vid 30°C och 37°C. En analys av den ribosomala statusen visade på en minskning av ribosomer aktiva i translation och en motsvarande ökning av fria 50S- och 30S-subenheter. Den oproportionerligt höga ökningen av fria 50S-subenheter, i relation till 30S-subenheter, indikerade att något var fel i monteringen av 30S-subenheten. RimP-proteinet återfanns lokaliserat till fria 30S-subenheter, och en ökning av omoget 16S ribosomalt RNA i en stam som saknar RimP stödjer dess roll i monteringen av 30S-subenheten. Rekonstrueringsexperiment In vitro har gett många värdefulla ledtrådar till hur 30S-subenheten monteras ihop. Genom att använda en nyligen utvecklad metod kunde vi undersöka hur mognadsfaktorerna Era, RimM och RimP påverkade monteringen av ribosomens 30S subenhet in vitro. Era ökade inkorporeringshastigheten av många av de ribosomala proteiner som inkorporeras sent i monteringen av 30S, medans RimM och RimP uppvisade mer specifika effekter. RimM ökade inkorporeringshastigheten för de ribosomala proteinerna S19 och S9, men dessutom inhiberade RimM inkorporeringen av de ribosomala proteinerna S13 och S12. RimP uppvisade den tydligaste effekten av de undersökta proteinerna genom att kraftigt öka 8 inkorporeringshastigheten för det ribosomala proteinet S12, och ökade även inkorporeringshastigheten för det ribosomala proteinet S5. En jämförelse av de två mognadsfaktorerna RbfA och RimP visade på strukturella likheter mellan RimP:s N-terminala domän och den enda domänen hos RbfA. RbfA är ett 15 kDa protein som upptäcktes som en hög-kopiesupressor av en dominant C23U-mutation i 16S ribosomalt RNA som leder till köld-känslighet hos E. coli. Ett antal kromosomala supressormutationer som ökade tillväxthastigheten för en mutant som saknar RbfA isolerades och de fem starkaste av dessa lokaliserades till rpsE genen som kodar för det ribosomala proteinet S5. Mutationerna gav upphov till aminosyra utbyten i tre positioner i S5: G87A, G87S, G91A, A127T och A127V. Förändringarna i S5 förbättrade translationen och processningen av 16S ribosomalt RNA i mutantensom saknar RbfA. Dessutom förbättrade mutationerna tillväxthastigheten hos C23U-mutanten vid 30, 37 och 44°C.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, Institutionen för molekylärbiologi, 2010. 54 p.
Keyword
Ribosome, 30S, rRNA, RimP, RbfA, C23U, rpsE
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-35890 (URN)978-91-7459-061-6 (ISBN)
Public defence
2010-10-01, 6A-103, NUS, Norrlands Universitetssjukhus, Umeå, 13:00 (English)
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Available from: 2010-09-10 Created: 2010-09-08 Last updated: 2010-09-21Bibliographically approved

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Nord, StefanBylund, Göran OWikström, P Mikael
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