Transfer-messenger RNA unfolds as it transits the ribosome

Wower, Iwona K.; Zwieb, Christian W; Wower, Jacek

doi:10.1261/rna.7269305

Cited by 21 publications

(21 citation statements)

References 30 publications

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“…The second state is the state after the release of EF-Tu, in which tmRNA has moved into the A site. The resulting density map, at 12-Å resolution, shows the tmRNA to be largely unstructured, in agreement with predictions from recent biochemical findings of Wower et al (17).…”

supporting

confidence: 89%

“…Another unsolved problem is the fate of tmRNA in the accommodated state. Wower et al (17) concluded from biochemical data that the tmRNA is partially unstructured after release of EF-Tu from the ribosome and accommodation of TLD, after insertion of the ORF into the mRNA channel. Again, a confirmation of this result by structural methods has thus far been missing, and it is as yet unclear how the binding or release of the SmpBs is coordinated with the unraveling of tmRNA.…”

mentioning

confidence: 99%

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Cryo-EM visualization of transfer messenger RNA with two SmpBs in a stalled ribosome

Kaur

Gillet

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

121

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In eubacterial translation, lack of a stop codon on the mRNA results in a defective, potentially toxic polypeptide stalled on the ribosome. Bacteria possess a specialized mRNA, called transfer messenger RNA (tmRNA), to rescue such a stalled system. tmRNA contains a transfer RNA (tRNA)-like domain (TLD), which enters the ribosome as a tRNA and places an ORF into the mRNA channel. This ORF codes for a signal marking the polypeptide for degradation and ends in a stop codon, leading to release of the faulty polypeptide and recycling of the ribosome. The binding of tmRNA to the stalled ribosome is mediated by small protein B (SmpB). By means of cryo-EM, we obtained a density map for the preaccommodated state of the tmRNA⅐SmpB⅐EF-Tu⅐70S ribosome complex with much improved definition for the tmRNA-SmpB complex, showing two SmpB molecules bound per ribosome, one toward the A site on the 30S subunit side and the other bound to the 50S subunit near the GTPase-associated center. tmRNA is strongly attached to the 30S subunit head by multiple contact sites, involving most of its pseudoknots and helices. The map clarifies that the TLD is located near helix 34 and protein S19 of the 30S subunit, rather than in the A site as tRNA for normal translation, so that the TLD is oriented toward the ORF. elongation factor Tu ͉ preaccommodated state ͉ rescue mechanism ͉ transtranslation ͉ transfer RNA-like domain

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supporting

confidence: 89%

mentioning

confidence: 99%

Cryo-EM visualization of transfer messenger RNA with two SmpBs in a stalled ribosome

Kaur

Gillet

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

121

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“…5A). As there are different structural intermediates during the folding (49) and function (19,50) of tmRNA, we wanted to confirm that ACAs in the single-strand region of the structure proposed previously by Zwieb et al (57) are more accessible to MazF cleavage than are double-strand ACAs.…”

Section: Correlation Of Mazf Homologues and The Absence Of Acas In Tmmentioning

confidence: 84%

Significant Bias against the ACA Triplet in the tmRNA Sequence of Escherichia coli K-12

et al. 2009

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The toxin MazF in Escherichia coli cleaves single-stranded RNAs specifically at ACA sequences. MazF overexpression virtually eliminates all cellular mRNAs to completely block protein synthesis. However, protein synthesis can continue on an mRNA that is devoid of ACA triplets. The finding that ribosomal RNAs remain intact in the face of complete translation arrest suggested a purpose for such preservation. We therefore examined the sequences of all transcribed RNAs to determine if there was any statistically significant bias against ACA. While ACA motifs are absent from tmRNA, 4.5S RNA, and seven of the eight 5S rRNAs, statistical analysis revealed that only for tmRNA was the absence nonrandom. The introduction of single-strand ACAs makes tmRNA highly susceptible to MazF cleavage. Furthermore, analysis of tmRNA sequences from 442 bacteria showed that the discrimination against ACA in tmRNAs was seen mostly in enterobacteria. We propose that the unusual bias against ACA in tmRNA may have coevolved with the acquisition of MazF.

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“…In addition, the putative unwinding of pseudo-knot structures PK2-PK4 (Kaur et al 2006) would require a significant input of free energy from a yet unidentified source. The observation that replacement of the stop codons at the end of the ORF of tmRNA with sense codons results in the synthesis of abnormally long-tag peptides (Wower et al 2005) shows that unwinding of the tmRNA secondary in the PK2-PK4 can take place. However, in this latter case the free energy for unwinding is provided by protein elongation, which supports the present and previous conclusions (Ivanov et al 2002;Shpanchenko et al 2005) that the putative unwinding of tmRNA takes place at a much later stage of trans-translation than tmRNA accommodation in the A site.…”

Section: Discussionmentioning

confidence: 99%

Structure probing of tmRNA in distinct stages of trans-translation

Ivanovа¹,

Lindell²,

Pavlov³

et al. 2007

RNA

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Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helper protein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work we used lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery at different steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reacted with Ala-tmRNA d EF-Tu d GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was probed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different transtranslation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we also analyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. We observed some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNA conformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA is in complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.

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Transfer-messenger RNA unfolds as it transits the ribosome

Cited by 21 publications

References 30 publications

Cryo-EM visualization of transfer messenger RNA with two SmpBs in a stalled ribosome

Cryo-EM visualization of transfer messenger RNA with two SmpBs in a stalled ribosome

Significant Bias against the ACA Triplet in the tmRNA Sequence of Escherichia coli K-12

Structure probing of tmRNA in distinct stages of trans-translation

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