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The flexible n-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor
Södertörn University, School of Life Sciences. Stockholm University.
Södertörn University, School of Life Sciences.
2007 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 365, no 3, 764-772 p.Article in journal (Refereed) Published
Abstract [en]

The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in 2 stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S (Delta L11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.

Place, publisher, year, edition, pages
2007. Vol. 365, no 3, 764-772 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:sh:diva-14242DOI: 10.1016/j.jmb.2006.10.065ISI: 000243561500021PubMedID: 17095013Scopus ID: 2-s2.0-33845743940OAI: oai:DiVA.org:sh-14242DiVA: diva2:467333
Available from: 2011-12-19 Created: 2011-12-19 Last updated: 2016-12-22Bibliographically approved
In thesis
1. The ribosome, stringent factor and the bacterial stringent response
Open this publication in new window or tab >>The ribosome, stringent factor and the bacterial stringent response
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The stringent response plays a significant role in the survival of bacteria during different environmental conditions. It is activated by the binding of stringent factor (SF) to stalled ribosomes that have an unacylated tRNA in the ribosomal A-site which leads to the synthesis of (p)ppGpp. ppGpp binds to the RNA polymerase, resulting in a rapid down-regulation of rRNA and tRNA transcription and up-regulation of mRNAs coding for enzymes involved in amino acid biosynthesis. The importance of the A-site and unacylated tRNA in the activation of SF was confirmed by chemical modification and subsequent primer extension experiments (footprinting experiments) which showed that binding of SF to ribosomes resulted in the protection of regions in 23S rRNA, the A-loop and helix 89 that are involved in the binding of the A-site tRNA. An in vitro assay showed that the ribosomal protein L11 and its flexible N-terminal part was important in the activation of SF. Interestingly the N-terminal part of L11 was shown to activate SF on its own and this activation was dependent on both ribosomes and an unacylated tRNA in the A-site. The N-terminal part of L11 was suggested to mediate an interaction between ribosome-bound SF and the unacylated tRNA in the A-site or interact with SF and the unacylated tRNA independently of each other. Footprinting experiments showed that SF bound to the ribosome protected bases in the L11 binding domain of the ribosome that were not involved in an interaction with ribosomal protein L11. The sarcin/ricin loop, in close contact with the L11 binding domain on the ribosome and essential for the binding and activation of translation elongation factors was also found to be protected by the binding of SF. Altogether the presented results suggest that SF binds to the factor-binding stalk of the ribosome and that activation of SF is dependent on the flexible N-terminal domain of L11 and an interaction of SF with the unacylated tRNA in the A-site of the 50S subunit.

Place, publisher, year, edition, pages
Stockholm: Wenner-Grens institut för experimentell biologi, 2007. 55 p.
Keyword
Ribosome, stringent factor, stringent response, tRNA, ribosomal protein L11, pppGpp
National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-31514 (URN)978-91-7155-414-7 (ISBN)
Public defence
2007-04-20, sal MA331, Alfred Nobels allé 7, Huddinge, 13:00
Opponent
Supervisors
Available from: 2016-12-22 Created: 2016-12-22 Last updated: 2016-12-22Bibliographically approved

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Citation style
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