Hydrodynamic properties of RNA: effect of multivalent cations on the sedimentation behavior of turnip yellow mosaic virus RNA

Eecen, H.G.; Dierendonck, Jan Hein van; Pleij, C.W.A.; Mandel, M.; Bosch, L.

doi:10.1021/bi00335a032

Cited by 15 publications

(7 citation statements)

References 41 publications

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“…An interaction of the protonated HP1 with the natural empty capsid, lacking a hole in the protein shell, was not observed, indicating that binding occurs at the interior of the protein shell. No (or reduced) binding was observed when magnesium ions were used instead of spermidine, despite the fact that magnesium is an important component of the TYMV virion (15).…”

Section: Discussionmentioning

confidence: 98%

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Protonation of non-Watson–Crick base pairs and encapsidation of turnip yellow mosaic virus RNA

Bink

Hellendoorn

Meulen

et al. 2002

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

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The 5 UTR of turnip yellow mosaic virus RNA contains two conserved hairpins with internal loops consisting of C⅐C and C⅐A mismatches. In this article, evidence is presented indicating that the 5 proximal hairpin functions as an encapsidation initiation signal. Extensive mutagenesis studies on this hairpin and sequencing of virus progeny showed a clear preference for C⅐C and C⅐A mismatches within the internal loop. The importance of these mismatches lies in their pH-dependent protonation and stable base pair formation. Encapsidation efficiency was found to be severely affected for several mutants lacking the protonatable mismatches in the internal loop of the 5 proximal hairpin. Furthermore, gel mobility-shift assays were performed with various RNA hairpins and empty capsids with a hole. Protonatable hairpins containing C⅐C and͞or C⅐A pairs were found to bind specifically to the interior of the protein shell under acidic conditions (pH 4.5) in the presence of spermidine. Based on these results we propose that this binding of protonated cytosines to the coat protein of turnip yellow mosaic virus may represent a new motif in RNA-protein interactions.T urnip yellow mosaic virus (TYMV) is a nonenveloped plant virus and the type member of the genus Tymovirus. The virion is an icosahedral particle with T ϭ 3 symmetry, and the protein shell consists of 180 identical subunits (1). The genomic RNA is 6,318 nt long and has an unusually high cytosine content (38%) and a rather low guanine content (18%). The genome carries three ORFs coding for the overlapping protein, which is thought to be involved in cell-to-cell transport, the RNAdependent RNA polymerase polyprotein, and the coat protein, which is expressed from a subgenomic messenger RNA. The 3Ј UTR has been shown to contain a tRNA-like structure whose function is still unclear (2).The 5Ј UTR of TYMV RNA is 90 nt long and has been shown, by means of structure probing and sequence comparison, to harbor two simple hairpins containing symmetric internal loops, which consist of C⅐C and C⅐A mismatches (ref. 3; see Fig. 1A). Under slightly acidic conditions these mismatches can form rather stable base pairs. NMR and UV melting experiments have confirmed that protonation of the C⅐C and C⅐A mismatches in hairpin 2 (HP2) stabilizes the structure when the pH is lowered from 7 to 5 (refs. 3 and 4; see Fig. 1B). A preliminary study of the possible function of the protonatable cytosines in the two 5Ј UTR hairpins suggested a dependence of TYMV on these nucleotides to establish a successful infection (5). A role for these hairpins in encapsidation was proposed. However, conclusions about the apparent requirement of C⅐C and͞or C⅐A mismatches were preliminary, as this study described one single mutant for which only a few revertants were recovered after passaging in Chinese cabbage. Moreover, this construct could not solve the question of whether an A⅐C pair instead of C⅐A was also allowed, because the chosen mutation rather forced the recovery of C⅐C and C⅐A mismatches through single ...

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Section: Discussionmentioning

confidence: 98%

“…Amounts of the polyamine spermidine up to as many as 400-500 molecules per virion (15) are believed to function in neutralizing the negative charge of the RNA phosphate residues. Furthermore, this polyamine was suggested to be instrumental in TYMV assembly (16), which is supported by our results.…”

Section: Discussionmentioning

confidence: 99%

Protonation of non-Watson–Crick base pairs and encapsidation of turnip yellow mosaic virus RNA

Bink

Hellendoorn

Meulen

et al. 2002

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

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“…The RNAs studied are all synthesized by in vitro transcription. In order to achieve reproducible ensembles, they are incubated in the buffer of interest for 24 h. Studies of the 6400-nt-long turnip yellow mosaic virus RNA (Eecen et al 1985) show that this treatment is necessary and sufficient to achieve highly reproducible hydrodynamic properties. We visualize the low-resolution secondary structure (branching pattern) of individual RNA molecules by cryo-electron microscopy (cryo-EM) in order to compute their ensemble sizes and shapes.…”

Section: Introductionmentioning

confidence: 99%

Visualizing large RNA molecules in solution

Gopal¹,

Zhou²,

Knobler³

et al. 2011

RNA

100

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Single-stranded RNAs (ssRNAs) longer than a few hundred nucleotides do not have a unique structure in solution. Their equilibrium properties therefore reflect the average of an ensemble of structures. We use cryo-electron microscopy to image projections of individual long ssRNA molecules and characterize the anisotropy of their ensembles in solution. A flattened prolate volume is found to best represent the shapes of these ensembles. The measured sizes and anisotropies are in good agreement with complementary determinations using small-angle X-ray scattering and coarse-grained molecular dynamics simulations. A long viral ssRNA is compared with shorter noncoding transcripts to demonstrate that prolate geometry and flatness are generic properties independent of sequence length and origin. The anisotropy persists under physiological as well as low-ionic-strength conditions, revealing a direct correlation between secondary structure asymmetry and 3D shape and size. We discuss the physical origin of the generic anisotropy and its biological implications.

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“…TYMV virions, on the other hand, contain nearly 1% by weight of polyamines, principally spermidine (Beer and Kosuge, 1970;Hirth and Givord, 1988). These neutralizing polyamines, estimated to be about 500 molecules per virion, along with an estimated 2000 Mg++ and Ca++ ions (Beer and Kosuge, 1970;Eecen et al, 1985), assume the roles that positively charged amino terminal polypeptides generally fulfill in plant viruses.…”

Section: Introductionmentioning

confidence: 99%

The RNA of turnip yellow mosaic virus exhibits icosahedral order

et al. 2005

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Difference electron density maps, based on structure factor amplitudes and experimental phases from crystals of wild-type turnip yellow mosaic virus and those of empty capsids prepared by freeze-thawing, show a large portion of the encapsidated RNA to have an icosahedral distribution. Four unique segments of base-paired, double-helical RNA, one to two turns in length, lie between 33-A and 101-A radius and are organized about either 2-fold or 5-fold icosahedral axes. In addition, single-stranded loops of RNA invade the pentameric and hexameric capsomeres where they contact the interior capsid surface. The remaining RNA, not seen in electron density maps, must serve as connecting links between these secondary structural elements and is likely icosahedrally disordered. The distribution of RNA observed crystallographically appears to be in agreement with models based on biochemical data and secondary structural analyses.

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Hydrodynamic properties of RNA: effect of multivalent cations on the sedimentation behavior of turnip yellow mosaic virus RNA

Cited by 15 publications

References 41 publications

Protonation of non-Watson–Crick base pairs and encapsidation of turnip yellow mosaic virus RNA

Protonation of non-Watson–Crick base pairs and encapsidation of turnip yellow mosaic virus RNA

Visualizing large RNA molecules in solution

The RNA of turnip yellow mosaic virus exhibits icosahedral order

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