Complementation of RNA Binding Site Mutations in MS2 Coat Protein Heterodimers

Peabody, David S.; Lim, Francis

doi:10.1093/nar/24.12.2352

Cited by 63 publications

(83 citation statements)

References 18 publications

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“…1D, solid line). This was in good agreement with previous analyses that proposed K D values of 0.4-3 nM using radiolabeled probes in filter binding studies as well as a K D value of z40 nM determined by tryptophan quenching analyses using unlabeled RNAs (Lim and Peabody 1994;Peabody and Lim 1996;Parrott et al 2000). Protein concentrations above z100 nM apparently led to the formation of larger complexes indicated by increased RNA binding, as previously proposed (Fig.…”

Section: Analysis Of Ms2bp/ms2 Binding By Nir Dye-labeled Rnassupporting

confidence: 93%

“…The MS2BP/MS2 association was used since binding had been investigated extensively using conventional approaches and this protein-RNA complex is widely used for tethering analyses (Lim and Peabody 1994;Peabody and Lim 1996;Bertrand et al 1998;Parrott et al 2000;Zhou and Reed 2003;Keryer-Bibens et al 2008). Moreover, the MS2BP/MS2 association requires formation of a stem-loop structure with at least one uridine in the loop that is essential for protein binding and two uridines involved in stem formation (Supplemental Fig.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, NIR-labeled UTP was used for in vitro transcription to validate how NIR dye modification affects both the structural properties and protein binding of NIR probes. By now, several mutations have been introduced in MS2BP that prevent phage assembly but retain RNA binding of MS2BP as a dimer with low nanomolar K D values (Peabody and Lim 1996;Peabody and AlBitar 2001;Lima et al 2006). Thus, the MS2BP/MS2 association was also expected to allow analyzing if NIRdye labeling of probes affects RNA-bindingdependent protein oligomerization.…”

Section: Introductionmentioning

confidence: 99%

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Near-infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the noncoding Y3-RNA

Köhn

Lederer²,

Wächter³

et al. 2010

RNA

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The analysis of protein-RNA association in vitro commonly involves radiolabeled in vitro transcribed RNAs. Nucleotides labeled with near-infrared (NIR) dyes provide promising alternatives for studying protein-RNA binding in vitro. However, it remained elusive whether random labeling of RNA probes by NIR dyes interferes with protein binding. Here, we demonstrate that infrared scanning allows the detection of randomly NIR-labeled RNA probes in the low femtomole range. The analyses of eight distinct protein-RNA complexes by electrophoretic mobility shift assay, filter binding, or UV crosslinking revealed that protein binding specificity remains unaffected by random NIR labeling. Accordingly, NIR probes allowed the rapid identification of the short noncoding Y3-RNA as a novel RNA target of ZBP1 (zipcode binding protein). Whereas binding of ZBP1 to the ACTB-zipcode and Y3 was exclusive, the protein formed a trimeric complex with the La protein and Y3. This was dissociated in the presence of Y5 RNA, resulting in the formation of ZBP1/Y3 and La/Y5 complexes. Hence, ZBP1 apparently resides in at least two distinct cellular RNPs: mRNA-containing mRNPs or Y3-containing yRNPs. In conclusion, our findings indicate that randomly labeled NIR probes provide a powerful tool for the rapid and sensitive analysis of protein-RNA binding in vitro. In contrast to radiolabeled RNAs, NIR probes remain stable for months, do not pose any safety considerations, and enable the significantly expedited analysis of experimental data due to fast read technologies available. The most prominent advantage of probes labeled by NIR dyes is the option to color-code distinct transcripts, allowing the unbiased identification of distinct protein-RNA complexes in one sample.

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Section: Analysis Of Ms2bp/ms2 Binding By Nir Dye-labeled Rnassupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Near-infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the noncoding Y3-RNA

Köhn

Lederer²,

Wächter³

et al. 2010

RNA

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“…After initial unsatisfactory results with this mutant, we fused a tandem dimer of the wild-type coat protein with an enhanced version of GFP. The fused dimer, MS2d, is known to be highly tolerant of structural perturbations and retains its in vivo functionality after insertion at various locations (23,24). We fused MS2d to the N terminus of GFPmut3 (25) and placed the fusion under the control of the tetracycline promoter P(LtetO-1) (26) in the vector K133 (based on the PROTET.E vector; Clontech).…”

Section: Methodsmentioning

confidence: 99%

RNA dynamics in live Escherichia coli cells

Golding

Cox

2004

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

272

290

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We describe a method for tracking RNA molecules in Escherichia coli that is sensitive to single copies of mRNA, and, using the method, we find that individual molecules can be followed for many hours in living cells. We observe distinct characteristic dynamics of RNA molecules, all consistent with the known life history of RNA in prokaryotes: localized motion consistent with the Brownian motion of an RNA polymer tethered to its template DNA, free diffusion, and a few examples of polymer chain dynamics that appear to be a combination of chain fluctuation and chain elongation attributable to RNA transcription. We also quantify some of the dynamics, such as width of the displacement distribution, diffusion coefficient, chain elongation rate, and distribution of molecule numbers, and compare them with known biophysical parameters of the E. coli system.T he bacterium Escherichia coli may well be the most thoroughly characterized model biological system. Much of its metabolism serves as the basic paradigm for DNA replication, RNA transcription, protein synthesis, and gene regulation (1, 2).Recently, techniques have become available that allow us to study central problems in genome organization and expression in individual living cells (3, 4), rather than rely on the averaged properties of large populations. These studies have added to our knowledge in two main areas: the heterogeneity among cells in a supposedly homogeneous population (5-7) and the spatiotemporal organization of macromolecules in the bacterial cell (8). The subcellular location of a variety of different proteins involved in cell division, such as the MinCDE family (9), are now known to be localized in special regions, as is the location of replicating chromosomes (10) and several different plasmids (11,12). Complexes of signaling proteins in Bacillus subtilis and Caulobacter crescentus have likewise been localized to the poles of dividing and differentiating cells (8, 13).To our knowledge, there are no published studies on the localization and timing of mRNA synthesis in living bacterial cells. Our knowledge of RNA transcription and dynamics comes from population studies, or in vitro studies with purified components (14-17). A rare glimpse into cell-to-cell variability in message number was provided by the studies of with Salmonella using whole fixed mounts. However, there is no information at the single-cell level in living cells on message localization: whether mRNA is free to move throughout the cytoplasm, whether movement is active or passive, and, for RNA molecules present in low copy numbers, how the actual number of molecules is a function of cell state. This last information is of particular interest because it bears directly on the question of the regulation of proteins known to be present in low copy numbers (for example, many of the proteins that act to switch genes on and off) and how the cell successfully regulates copy number in the presence of considerable extrinsic and intrinsic noise (see ref. 7 for a review).Here we describe a metho...

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“…Moreover, monomers would be inactive in the assay and occupy LexA operators unproductively. We therefore created a new strain, YBZ1, carrying a tandem, head-to-tail dimer of MS2 coat protein fused to a single LexA monomer (Fig 5B;Peabody and Lim 1996). In the crystal structure of an MS2 coat protein dimer, the N and C termini are relatively close to one another (Valegard et al 1994); a nine-amino-acid linker was introduced to enable flexibility in orienting the two MS2 segments.…”

Section: An Improved Yeast Strain For Library Screeningmentioning

confidence: 99%

RNA–protein interactions in the yeast three-hybrid system: Affinity, sensitivity, and enhanced library screening

Hook

Bernstein²,

Zhang³

et al. 2004

RNA

114

116

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The yeast three-hybrid system has become a useful tool in analyzing RNA-protein interactions. An RNA sequence is tested in combination with an RNA-binding protein linked to a transcription activation domain (AD). A productive RNA-protein interaction activates a reporter gene in vivo. The system has been used to test candidate RNA-protein pairs, to isolate mutations in each interacting partner, and to identify proteins that bind a given RNA sequence. However, the relationship between reporter gene activation and in vitro affinity of an RNA-protein interaction has not been examined

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Complementation of RNA Binding Site Mutations in MS2 Coat Protein Heterodimers

Cited by 63 publications

References 18 publications

Near-infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the noncoding Y3-RNA

Near-infrared (NIR) dye-labeled RNAs identify binding of ZBP1 to the noncoding Y3-RNA

RNA dynamics in live Escherichia coli cells

RNA–protein interactions in the yeast three-hybrid system: Affinity, sensitivity, and enhanced library screening

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