Domain III of the <i>T. thermophilus</i> 23S rRNA folds independently to a near-native state

Athavale, Shreyas S.; Gossett, J. Jared; Hsiao, Chiaolong; Bowman, Jessica C.; O’Neill, Eric B.; Hershkovitz, Eli; Preeprem, Thanawadee; Hud, Nicholas V.; Wartell, Roger M.; Harvey, Stephen C.; Williams, Loren Dean

doi:10.1261/rna.030692.111

Cited by 21 publications

(39 citation statements)

References 45 publications

(66 reference statements)

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“…Some RNAs, e.g. , tRNA, RNase P, the Tetrahymena thermophila group I intron P4–P6 domain, and domain III of the T. thermophilus 23S rRNA, show significant Mg 2+ -dependence of SHAPE reactivities [25]–[29]. STMV RNA is essentially an mRNA, so its folding is not necessarily expected to be dependent on Mg 2+ .…”

Section: Resultsmentioning

confidence: 99%

“…SHAPE probing of STMV RNA was performed as described in [29]. Five 20-nt long DNA primers were used to primer reverse transcription reactions.…”

Section: Methodsmentioning

confidence: 99%

“…This procedure has been described in detail in Athavale et al [29]. Briefly, this involved (1) aligning the traces to one another, (2) calculating and subtracting the baseline, (3) locating the peaks, (4) quantifying the area of each peak, (5) correcting for signal decay, (6) subtracting the background, and (7) normalizing.…”

Section: Methodsmentioning

confidence: 99%

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In Vitro Secondary Structure of the Genomic RNA of Satellite Tobacco Mosaic Virus

et al. 2013

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Satellite tobacco mosaic virus (STMV) is a T = 1 icosahedral virus with a single-stranded RNA genome. It is widely accepted that the RNA genome plays an important structural role during assembly of the STMV virion. While the encapsidated form of the RNA has been extensively studied, less is known about the structure of the free RNA, aside from a purported tRNA-like structure at the 3′ end. Here we use selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to examine the secondary structure of in vitro transcribed STMV RNA. The predicted secondary structure is unusual in the sense that it is highly extended, which could be significant for protecting the RNA from degradation. The SHAPE data are also consistent with the previously predicted tRNA-like fold at the 3′ end of the molecule, which is also known to hinder degradation. Our data are not consistent with the secondary structure proposed for the encapsidated RNA by Schroeder et al., suggesting that, if the Schroeder structure is correct, either the RNA is packaged as it emerges from the replication complex, or the RNA undergoes extensive refolding upon encapsidation. We also consider the alternative, i.e., that the structure of the encapsidated STMV RNA might be the same as the in vitro structure presented here, and we examine how this structure might be organized in the virus. This possibility is not rigorously ruled out by the available data, so it remains open to examination by experiment.

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Section: Resultsmentioning

confidence: 99%

“…SHAPE probing of STMV RNA was performed as described in [29]. Five 20-nt long DNA primers were used to primer reverse transcription reactions.…”

Section: Methodsmentioning

confidence: 99%

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In Vitro Secondary Structure of the Genomic RNA of Satellite Tobacco Mosaic Virus

et al. 2013

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“…SHAPE data were processed as described earlier (33). Several nucleotides were excluded from SHAPE analysis because the reverse transcriptase gave high background termination in the (−) NMIA reactions.…”

Section: Methodsmentioning

confidence: 99%

Molecular paleontology: a biochemical model of the ancestral ribosome

Hsiao

Lenz

Peters

et al. 2013

Nucleic Acids Research

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Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.

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“…STMV RNA was imaged in 5 mM Mg 2+ to reflect the divalent cation concentration in vivo. We had previously shown that Mg 2+ does not affect the SHAPE-restrained secondary structure of STMV RNA (Athavale et al 2013), but it can induce tertiary interactions in other RNA molecules (Athavale et al 2012), making them more compact when visualized by cryo-EM (Gopal et al 2012). The yeast RNA was imaged in the absence of Mg 2+ to minimize the possibility of such tertiary interactions; that is, we omitted Mg 2+ from the yeast sample in an effort to make the control molecule as extended as possible.…”

Section: Rna Synthesis and Purificationmentioning

confidence: 99%

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

Garmann

Gopal

Athavale

et al. 2015

RNA

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The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2 ′ -hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.

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Domain III of the T. thermophilus 23S rRNA folds independently to a near-native state

Cited by 21 publications

References 45 publications

In Vitro Secondary Structure of the Genomic RNA of Satellite Tobacco Mosaic Virus

In Vitro Secondary Structure of the Genomic RNA of Satellite Tobacco Mosaic Virus

Molecular paleontology: a biochemical model of the ancestral ribosome

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

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