Visualizing tmRNA Entry into a Stalled Ribosome

  title={Visualizing tmRNA Entry into a Stalled Ribosome},
  author={Mikel Valle and Reynald Gillet and Sukhjit Kaur and Anke Henne and Venkatraman Ramakrishnan and Joachim Frank},
  pages={127 - 130}
Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, an ∼300-nucleotide-long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation then switches from the defective message to a short open reading frame on tmRNA that tags the defective nascent peptide chain for degradation. However, the mechanism by which tmRNA can enter and move through the ribosome is unknown. We present a cryo–electron microscopy study at ∼13 to 15 angstroms of the entry of… 
tmRNA on its way through the ribosome
Determination of the structure of tmRNA and SmpB in complex with the ribosome, at the stage when translation has resumed on tmRNAs, has provided an increased understanding and unique insights into the complex mechanism of template switching on the Ribosome and Sm pB-driven selection of the correct reading frame on t mRNA’s ORF.
Structural features of the tmRNA-ribosome interaction.
Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation, and it is exploited to examine the t mRNA structure using chemical probing and cryo-electron microscopy tomography.
Structures of tmRNA and SmpB as they transit through the ribosome
Four cryo-EM structures of the ribosome during trans-translation are presented and light is shed on the movements of the tmRNA-SmpB complex in theribosome, from its delivery by the elongation factor EF-Tu to its passage through the Ribosomal A and P sites after the opening of the B1 bridges.
Cryo-EM visualization of transfer messenger RNA with two SmpBs in a stalled ribosome
A density map for the preaccommodated state of the tmRNA·SmpB·EF-Tu·70S ribosome complex is obtained with much improved definition for the TLD, showing two SmpB molecules bound per ribosomes, one toward the A site on the 30S sub unit side and the other bound to the 50S subunit near the GTPase-associated center.
Accommodation of tmRNA-SmpB into stalled ribosomes: a cryo-EM study.
Using a construct containing the tRNA-like domain as well as the extended helix H2 of tm RNA, a cryo-electron microscopy study of the process of accommodation is presented and suggests that the larger movement required to resume translation on a tmRNA internal open reading frame starts during accommodation.
The tmRNA ribosome-rescue system.
Dial tm for rescue: tmRNA engages ribosomes stalled on defective mRNAs.
Visualizing the transfer-messenger RNA as the ribosome resumes translation
This study obtained the first structure of an in vivo‐formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site, and built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tm RNA.
The tmRNA system for translational surveillance and ribosome rescue.
Structural and biochemical studies suggest mechanisms that keep tmRNA from interrupting normal translation and target ribosomes stalled with very short 3' mRNA extensions.


Emerging views on tmRNA‐mediated protein tagging and ribosome rescue
An updated model of tmRNA mediated protein tagging and ribosome rescue in bacteria is presented and the importance of bacterial tmRNAs for survival, growth under stress, and pathogenesis is discussed.
The SsrA–SmpB system for protein tagging, directed degradation and ribosome rescue
The structural, functional and phylogenetic properties of this unique RNA and its associated factors are reviewed, and the intracellular proteases that act to degrade the proteins tagged by this system are also discussed.
Kinetic parameters for tmRNA binding to alanyl-tRNA synthetase and elongation factor Tu from Escherichia coli.
Kinetic analysis of the aminoacylation of tmRNAs indicates that tmRNA has both a lower affinity and a lower turnover number than cognate tRNA(Ala) for alanyl-tRNA synthetase, resulting in a 75-fold lower k(cat)/K(M) value, which can be interpreted to suggest that additional factors facilitate tm RNA binding to ribosomes.
Simultaneous and functional binding of SmpB and EF-Tu-TP to the alanyl acceptor arm of tmRNA.
Kinetic analysis of functioning combined with band-shift experiments and structural probing demonstrate, that tmRNA may indeed form a multimeric complex with SmpB, S1 and EF-Tu-GTP, which leads to a considerably enhanced efficiency of the initial steps of trans-translation.
Structure of the 30S ribosomal subunit
The crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 Å resolution, is reported, which will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.
Visualization of elongation factor Tu on the Escherichia coli ribosome
Electron cryomicroscopy and angular reconstitution are used to visualize directly the kirromycin-stalled ternary complex in the A site of the 70S ribosome of Escherichia coli and three-dimensional reconstruction at 18 Å resolution shows the ternaries spanning the inter-subunit space with the acceptor domain of the tRNA reaching into the decoding centre.
Resuming translation on tmRNA: a unique mode of determining a reading frame
It is found that the ribosome can reach and use the extreme 3′ terminal codon of the defective mRNA prior to switching and the first triplet to be translated in tmRNA (the resume codon) is determined at two levels.
The biological roles of trans-translation.