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Unstructured 5'-tails act through ribosome standby to override inhibitory structure at ribosome binding sites.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
2018 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 8, p. 4188-4199Article in journal (Refereed) Published
Abstract [en]

Initiation is the rate-limiting step in translation. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. The ribosome standby model of de Smit and van Duin, based on studies of the MS2 phage coat cistron, proposed how high translation rates can be reconciled with stable, inhibitory structures at an RBS. Here, we revisited the coat protein system and assessed the translation efficiency from its sequestered RBS by introducing standby mutations. Further experiments with gfp reporter constructs assessed the effects of 5-tails-as standby sites-with respect to length and sequence contributions. In particular, combining in vivo and in vitro assays, we can show that tails of CA-dinucleotide repeats-and to a lesser extent, AU-repeats-dramatically increase translation rates. Tails of increasing length reach maximal rate-enhancing effects at 16-18 nucleotides. These standby tails are single-stranded and do not exert their effect by structure changes in the neighboring RBS stem-loop. In vitro translation and toeprinting assays furthermore demonstrate that standby effects are exerted at the level of translation initiation. Finally, as expected, destabilizing mutations within the coat RBS indicate an interplay with the effects of standby tails.

Place, publisher, year, edition, pages
Oxford University Press, 2018. Vol. 46, no 8, p. 4188-4199
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-343078DOI: 10.1093/nar/gky073ISI: 000431895800039PubMedID: 29420821OAI: oai:DiVA.org:uu-343078DiVA, id: diva2:1185474
Funder
Swedish Research Council, VR 621-2010-5233Available from: 2018-02-25 Created: 2018-02-25 Last updated: 2018-08-10Bibliographically approved
In thesis
1. Ribosome standby sites and other structural aspects of translation initiation regions in Escherichia coli
Open this publication in new window or tab >>Ribosome standby sites and other structural aspects of translation initiation regions in Escherichia coli
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Translation initiation, which is rate-limiting in protein synthesis, is often the step at which regulation occurs. Here, we investigated several mechanisms of translation initiation in Escherichia coli, including their control. First, we showed that translation of the transcriptional regulator CsgD is inhibited by two sRNAs through a direct antisense mechanism.

In some bacterial mRNAs, the ribosome binding site (RBS) is sequestered in a stable structure, which generally generates very low protein output. Yet, these mRNAs are often efficiently translated, which suggested the requirement for “ribosome standby sites”. Here, we investigated the structure and sequence features of an effective standby site using plasmid-borne GFP reporter constructs, and showed that relatively short, single-stranded regions near a structurally sequestered RBS can profoundly increase translation rates. Both the length and the sequence of these single-stranded regions are important for standby site efficiency, and the standby site needs to be single-stranded. This work serves as a proof-of-principle study of the ribosome standby model.

To investigate the sequence-dependency of standby sites further, we used an unbiased approach, creating plasmid libraries containing millions of different standby sites in the same reporter plasmid as before. Cells were sorted by fluorescence according to translational levels, and standby sites analyzed by deep sequencing. This analysis showed that efficient standby sites have a low GC-content and rarely contain Shine-Dalgarno sequences. Additionally, nucleotides near the 3’-border of the standby region affect translation efficiency more than those closer to the 5’-end. Mutational and structure-probing experiments are planned to verify these findings.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 58
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1636
Keywords
Ribosome standby sites, translation initiation, mRNA structure, bacteria
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-343082 (URN)978-91-513-0249-2 (ISBN)
Public defence
2018-04-13, Room A1:111a, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2018-03-23 Created: 2018-02-25 Last updated: 2018-04-24

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Romilly, CedricWagner, Gerhart E. H.

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