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Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli
Södertörns högskola, Institutionen för livsvetenskaper. Stockholm University.
Södertörns högskola, Institutionen för livsvetenskaper.
2005 (Engelska)Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 272, nr 3, s. 685-695Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However, tRNA-saturation curves, performed at two different ribosome/stringent factor ratios and filter-binding assays, did not support this hypothesis.

Ort, förlag, år, upplaga, sidor
2005. Vol. 272, nr 3, s. 685-695
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:sh:diva-14393DOI: 10.1111/j.1742-4658.2004.04502.xISI: 000227359400006PubMedID: 15670150Scopus ID: 2-s2.0-13444252553OAI: oai:DiVA.org:sh-14393DiVA, id: diva2:469126
Tillgänglig från: 2011-12-22 Skapad: 2011-12-21 Senast uppdaterad: 2017-12-08Bibliografiskt granskad
Ingår i avhandling
1. The ribosome, stringent factor and the bacterial stringent response
Öppna denna publikation i ny flik eller fönster >>The ribosome, stringent factor and the bacterial stringent response
2007 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: Wenner-Grens institut för experimentell biologi, 2007. s. 55
Nyckelord
Ribosome, stringent factor, stringent response, tRNA, ribosomal protein L11, pppGpp
Nationell ämneskategori
Cellbiologi
Identifikatorer
urn:nbn:se:sh:diva-31514 (URN)978-91-7155-414-7 (ISBN)
Disputation
2007-04-20, sal MA331, Alfred Nobels allé 7, Huddinge, 13:00
Opponent
Handledare
Tillgänglig från: 2016-12-22 Skapad: 2016-12-22 Senast uppdaterad: 2016-12-22Bibliografiskt granskad

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Knutsson Jenvert, Rose-MarieHolmberg Schiavone, Lovisa

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