Lead(II) as a probe for investigating RNA structure in vivo

Lindell, Magnus; Romby, Pascale; Wagner, E. Gerhart H.

doi:10.1017/s1355838201020416

Cited by 72 publications

(59 citation statements)

References 30 publications

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“…Lead(II) probing of tmRNA in vivo has corroborated this model and suggests similar tmRNA folding in vitro and in vivo (Knudsen et al 2001;Lindell et al 2002).…”

Section: Introductionmentioning

confidence: 52%

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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“…Lead(II) probing of tmRNA in vivo has corroborated this model and suggests similar tmRNA folding in vitro and in vivo (Knudsen et al 2001;Lindell et al 2002).…”

Section: Introductionmentioning

confidence: 52%

Structure probing of tmRNA in distinct stages of trans-translation

Ivanovа¹,

Lindell²,

Pavlov³

et al. 2007

RNA

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“…2). Interactions were probed with lead (II) acetate, which cleaves within single-stranded and dynamic regions of RNA (Brunel and Romby 2000;Lindell et al 2002), and RNase A, which cleaves after unpaired Cs and Us (Raines 1998). We used 59-and 39-end-labeled RNA to resolve interactions with the 39 and 59 regions of the repeat, respectively ( Fig.…”

Section: Mapping the Cas6-crispr Repeat Rna Binding Sitementioning

confidence: 99%

Binding and cleavage of CRISPR RNA by Cas6

Carte¹,

Pfister²,

Compton³

et al. 2010

RNA

146

158

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The CRISPR-Cas system provides many prokaryotes with acquired resistance to viruses and other mobile genetic elements. The core components of this defense system are small, host-encoded prokaryotic silencing (psi)RNAs and Cas (CRISPR-associated) proteins. Invader-derived sequences within the psiRNAs guide Cas effector proteins to recognize and silence invader nucleic acids. Critical for CRISPR-Cas defense is processing of the psiRNAs from the primary transcripts of the host CRISPR (clustered regularly interspaced short palindromic repeat) locus. Cas6, a previously identified endoribonuclease present in a wide range of prokaryotes with the CRISPR-Cas system, binds and cleaves within the repeat sequences that separate the individual invader targeting elements in the CRISPR locus transcript. In the present study, we investigated several key aspects of the mechanism of function of Cas6 in psiRNA biogenesis. RNA footprinting reveals that Pyrococcus furiosus Cas6 binds to a 7-nt (nucleotide) sequence near the 59 end of the CRISPR RNA repeat sequence, 14 nt upstream of the Cas6 cleavage site. In addition, analysis of the cleavage activity of P. furiosus Cas6 proteins with mutations at conserved residues suggests that a triad comprised of Tyr31, His46, and Lys52 plays a critical role in catalysis, consistent with a possible general acid-base RNA cleavage mechanism for Cas6. Finally, we show that P. furiosus Cas6 remains stably associated with its cleavage products, suggesting additional roles for Cas6 in psiRNA biogenesis.

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“…In Vivo Lead(II) Acetate Probing of RNA-The protocol used has essentially been described previously (37). P. fluorescens cells were grown at 30°C in NYB.…”

Section: Construction Of Rsmy Mutant Alleles Andmentioning

confidence: 99%

A Repeated GGA Motif Is Critical for the Activity and Stability of the Riboregulator RsmY of Pseudomonas fluorescens

Valverde

Lindell

Wagner

et al. 2004

Journal of Biological Chemistry

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The riboregulator RsmY of Pseudomonas fluorescens strain CHA0 is an example of small regulatory RNAs belonging to the global Rsm/Csr regulatory systems controlling diverse cellular processes such as glycogen accumulation, motility, or formation of extracellular products in various bacteria. By binding multiple molecules of the small regulatory protein RsmA, RsmY relieves the negative effect of RsmA on the translation of several target genes involved in the biocontrol properties of strain CHA0. RsmY and functionally related riboregulators have repeated GGA motifs predicted to be exposed in single-stranded regions, notably in the loops of hairpins. The secondary structure of RsmY was corroborated by in vivo cleavage with lead acetate. RsmY mutants lacking three or five (out of six) of the GGA motifs showed reduced ability to derepress the expression of target genes in vivo and failed to bind the RsmA protein efficiently in vitro. The absence of GGA motifs in RsmY mutants resulted in reduced abundance of these transcripts and in a shorter half-life (<6 min as compared with 27 min for wild type RsmY). These results suggest that both the interaction of RsmY with RsmA and the stability of RsmY strongly depend on the GGA repeats and that the ability of RsmY to interact with small regulatory proteins such as RsmA may protect this RNA from degradation.In prokaryotes, an increasing number of small, noncoding RNAs has been described; many of them have been predicted by bioinformatic approaches in Escherichia coli, raising their number to Ͼ60 in this organism (1-3). Riboregulators (i.e. small, untranslated, regulatory RNAs) regulate gene expression at a post-transcriptional level and can be grouped into two main classes. A first class displays antisense base-pairing activity, which can regulate target mRNA translation or stability; DsrA (87 nt) 1 and RyhB (90 nt) are two such examples in E. coli. Different domains of DsrA interact with rpoS and hns mRNAs, activating rpoS but inhibiting hns translation (4), whereas RyhB negatively regulates the expression of mRNAs coding for proteins involved in iron storage (5). DsrA, RyhB, and several other antisense riboregulators require the RNA chaperone Hfq for their activity (6, 7).A second group of riboregulators includes molecules that antagonize small, regulatory, mRNA-binding proteins of the CsrA type. In E. coli, CsrA (for carbon storage regulator) can act as a negative regulator of glgCAP and cstA target mRNAs, or as a positive regulator of fhlDC mRNA (8 -10). By binding to the leader region of target mRNAs, CsrA (a dimeric protein with a 61-aa subunit) can block translation and destabilize the mRNA (8). The regulatory functions of CsrA are counteracted by the expression of two riboregulators, CsrB (360 nt) and CsrC (257 nt) (11, 12). Both RNAs can bind several molecules of CsrA, thus removing this protein from its target mRNA sites and thereby relieving post-transcriptional regulation (11, 12). In the biocontrol organism Pseudomonas fluorescens CHA0, a similar system controls t...

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Lead(II) as a probe for investigating RNA structure in vivo

Cited by 72 publications

References 30 publications

Structure probing of tmRNA in distinct stages of trans-translation

Structure probing of tmRNA in distinct stages of trans-translation

Binding and cleavage of CRISPR RNA by Cas6

A Repeated GGA Motif Is Critical for the Activity and Stability of the Riboregulator RsmY of Pseudomonas fluorescens

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