Diagenode

The ribosomal protein S1-dependent standby site in mRNA consists ofa single-stranded region and a 5' structure element.


Romilly Cédric et al.

In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient mRNAs, we provide a thorough characterization of the standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30∆S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 mRNA (∼100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.

Tags
D-Plex RNA-seq kit

Share this article

Published
August, 2019

Source

Products used in this publication

  • D-Plex Total RNA-seq Kit for Illumina
    C05030031
    D-Plex Total RNA-seq Kit for Illumina

イベント

  • FASEB Biological Methylation: Fundamental Mechanisms
    Porto, Portugal
    Jul 28-Aug 1, 2024
 すべてのイベントを見る

 


       Site map   |   Contact us   |   Conditions of sales   |   Conditions of purchase   |   Privacy policy