A thermodynamic analysis of the sequence-specific binding of RNA by bacteriophage MS2 coat protein

Johansson, Hans; Dertinger, Dagmar; LeCuyer, Karen A.; Behlen, Linda S.; Greef, Charles; Uhlenbeck, Olke C.

doi:10.1073/pnas.95.16.9244

Cited by 92 publications

(88 citation statements)

References 47 publications

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“…Other codon-directed mutagenesis experiments confirmed earlier results showing that the identity of Asn-87 was for specific binding of MS2 RNA and for discrimination of Qb RNA binding (Spingola and Peabody, 1997). Intensively were studied also crystal structures of complexes between recombinant MS2 capsids with mutations and RNA operator sequences (Valegârd et al, 1997;Johansson et al, 1998;Helgstrand et al, 2002;Horn et al, 2004). The conversion of Asn-87 to Ala improved the ability of purine-RNAs (GA operator type) to bind MS2 coat protein, but binding to pyrimidine-RNAs (MS2 operator type) still remained tighter (Johansson et al, 1998).…”

Section: Introductionsupporting

confidence: 68%

“…Intensively were studied also crystal structures of complexes between recombinant MS2 capsids with mutations and RNA operator sequences (Valegârd et al, 1997;Johansson et al, 1998;Helgstrand et al, 2002;Horn et al, 2004). The conversion of Asn-87 to Ala improved the ability of purine-RNAs (GA operator type) to bind MS2 coat protein, but binding to pyrimidine-RNAs (MS2 operator type) still remained tighter (Johansson et al, 1998). The authors also appointed the possible role of some other amino acids in the complex formation and particularly of Ser-52, Asn-55 and Lys-57.…”

Section: Introductionmentioning

confidence: 99%

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In Vivo Packaging of mRNA in Yeast-Produced Bacteriophage GA Derived Virus-Like Particles

Argule

Cielēns

Renhofa

et al. 2017

Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences.

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Section: Introductionsupporting

confidence: 68%

Section: Introductionmentioning

confidence: 99%

In Vivo Packaging of mRNA in Yeast-Produced Bacteriophage GA Derived Virus-Like Particles

Argule

Cielēns

Renhofa

et al. 2017

Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences.

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“…We suspect that the MS2d-GFP proteins bound to the RNA molecule might ''immortalize'' it, i.e., prevent or at least considerably slow down its degradation. The protein͞RNA off-rate for the binding site sequence in use is on the order of many hours (52)(53)(54), which suggests that MS2d-GFP might not be displaced by the various RNA-degrading enzymes in the cell.…”

Section: Results and Discussion Ms2-rna Complexes In Vitromentioning

confidence: 99%

RNA dynamics in live Escherichia coli cells

Golding

Cox

2004

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

273

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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“…Pum binds to the PRE with a dissociation constant in the low nano-molar range (Zamore et al 1997(Zamore et al , 1999. To strengthen this interaction, we utilized a modified MS2 coat protein and binding site interaction with an order of magnitude stronger binding (Lim et al 1994;Johansson et al 1998). Two reporters were used in this analysis: RnLuc MS2 pA-the 3 ′ poly(A) tail was generated using efficient cleavage/polyadenylation elements, whereas in the RnLuc MS2 HSL reporter, a nonadenylated 3 ′ end was generated by the HSL (Fig.…”

Section: The Rbd Of Pumilio Enhances Deadenylation Dependent On the Pmentioning

confidence: 99%

The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation

Weidmann

Raynard

Blewett

et al. 2014

RNA

117

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PUF proteins are potent repressors that serve important roles in stem cell maintenance, neurological processes, and embryonic development. These functions are driven by PUF protein recognition of specific binding sites within the 3 ′ untranslated regions of target mRNAs. In this study, we investigated mechanisms of repression by the founding PUF, Drosophila Pumilio, and its human orthologs. Here, we evaluated a previously proposed model wherein the Pumilio RNA binding domain (RBD) binds Argonaute, which in turn blocks the translational activity of the eukaryotic elongation factor 1A. Surprisingly, we found that Argonautes are not necessary for repression elicited by Drosophila and human PUFs in vivo. A second model proposed that the RBD of Pumilio represses by recruiting deadenylases to shorten the mRNA's polyadenosine tail. Indeed, the RBD binds to the Pop2 deadenylase and accelerates deadenylation; however, this activity is not crucial for regulation. Rather, we determined that the poly(A) is necessary for repression by the RBD. Our results reveal that poly(A)-dependent repression by the RBD requires the poly(A) binding protein, pAbp. Furthermore, we show that repression by the human PUM2 RBD requires the pAbp ortholog, PABPC1. Pumilio associates with pAbp but does not disrupt binding of pAbp to the mRNA. Taken together, our data support a model wherein the Pumilio RBD antagonizes the ability of pAbp to promote translation. Thus, the conserved function of the PUF RBD is to bind specific mRNAs, antagonize pAbp function, and promote deadenylation.

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A thermodynamic analysis of the sequence-specific binding of RNA by bacteriophage MS2 coat protein

Cited by 92 publications

References 47 publications

In Vivo Packaging of mRNA in Yeast-Produced Bacteriophage GA Derived Virus-Like Particles

In Vivo Packaging of mRNA in Yeast-Produced Bacteriophage GA Derived Virus-Like Particles

RNA dynamics in live Escherichia coli cells

The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation

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