Mutagenesis of a stacking contact in the MS2 coat protein-RNA complex.

LeCuyer, Karen A.; Behlen, Linda S.; Uhlenbeck, Olke C.

doi:10.1002/j.1460-2075.1996.tb01076.x

Cited by 59 publications

(46 citation statements)

References 35 publications

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“…3, 8). Analogous data have been reported with certain other complexes: For example, alanine substitution of a stacking tyrosine in the MS2 coat protein-RNA complex reduces binding 160-fold, while substitution of a phenylalanine in the U1A-RNA complex is reported to reduce binding 10,000-fold (LeCuyer et al 1996;Nolan et al 1999). We suggest that the abundance of consecutive stacking interactions in the FBF-2-RNA complex mitigates the effects of single mutations, and local plasticity allows substitutions of stacking side chains with reduced effect on binding affinity.…”

Section: Discussionsupporting

confidence: 73%

Stacking interactions in PUF–RNA complexes

Koh¹,

Wang²,

Qiu³

et al. 2011

RNA

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Stacking interactions between amino acids and bases are common in RNA-protein interactions. Many proteins that regulate mRNAs interact with single-stranded RNA elements in the 39 UTR (39-untranslated region) of their targets. PUF proteins are exemplary. Here we focus on complexes formed between a Caenorhabditis elegans PUF protein, FBF, and its cognate RNAs. Stacking interactions are particularly prominent and involve every RNA base in the recognition element. To assess the contribution of stacking interactions to formation of the RNA-protein complex, we combine in vivo selection experiments with site-directed mutagenesis, biochemistry, and structural analysis. Our results reveal that the identities of stacking amino acids in FBF affect both the affinity and specificity of the RNA-protein interaction. Substitutions in amino acid side chains can restrict or broaden RNA specificity. We conclude that the identities of stacking residues are important in achieving the natural specificities of PUF proteins. Similarly, in PUF proteins engineered to bind new RNA sequences, the identity of stacking residues may contribute to ''target'' versus ''off-target'' interactions, and thus be an important consideration in the design of proteins with new specificities.

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Section: Discussionsupporting

confidence: 73%

Stacking interactions in PUF–RNA complexes

Koh¹,

Wang²,

Qiu³

et al. 2011

RNA

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“…We found that Tyr at position 122 in M.CviBIII is the site of modification in the photo-cross-linking reaction. Very high photo-cross-linking yields were also reported for the RNA-binding domain of the U1A spliceosomal protein (20) and the bacteriophage MS2 coat protein (21), where three-dimensional structures of the protein-RNA complexes are known (34,35). In these complexes a Tyr residue is found in a -stacking arrangement with a cytosine residue, and replacement of these cytosine residues by 5-iodouracil resulted in 67-90% photo-cross-linking yields.…”

Section: Dna Binding Andmentioning

confidence: 86%

Identification of the Binding Site for the Extrahelical Target Base in N 6-Adenine DNA Methyltransferases by Photo-cross-linking with Duplex Oligodeoxyribonucleotides Containing 5-Iodouracil at the Target Position

Holz

Dank

Eickhoff

et al. 1999

Journal of Biological Chemistry

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DNA methyltransferases (Mtases) 1 are a biologically important class of enzymes that catalyze the transfer of the activated methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to the N-6 nitrogen of adenine and the N-4 nitrogen of cytosine or the C-5 carbon of cytosine within their DNA recognition sequences (1). As DNA methylation is a postreplicative process that depends on the presence or regulation of DNA Mtases, a particular DNA sequence may exist in its fully methylated, its unmethylated, or transiently in its hemimethylated form. Thus, DNA methylation can be regarded as an increase of the information content of DNA (2), which serves a wide variety of biological functions, including protection of the host genome from endogenous restriction endonucleases, DNA mismatch repair after replication, regulation of gene expression and DNA replication, embryonic development, genomic imprinting, and X-chromosome inactivation (3-5). Furthermore, it plays a role in carcinogenesis (6). Three-dimensional structures are available for the C 5 -cytosine DNA Mtases M.HhaI (7) and M.HaeIII (8) in complex with DNA. Both enzymes consist of two domains forming a positively charged cleft, which accommodates the DNA. However, the most striking feature of these protein-DNA complexes is that the target cytosines are completely rotated out of the DNA double helix and placed in a pocket within the cofactor binding domains, where catalysis takes place. In addition, the structures of the N 6 -adenine DNA Mtases M.TaqI (9) and the N 4 -cytosine DNA Mtase M.PvuII (10) in the absence of DNA were reported. These N 6 -adenine and N 4 -cytosine DNA Mtases (N-DNA Mtases) also have a bilobal structure forming a positively charged cleft. Modeling B-DNA in this cleft showed that the distance between the target base and the bound AdoMet is too large for a direct methyl group transfer, and a base flipping mechanism as observed for the C 5 -cytosine DNA Mtases was postulated. Biochemical evidence for a base flipping mechanism of the N 6 -adenine DNA Mtases M.EcoRI (11) and M.TaqI (12) was obtained using duplex oligodeoxyribonucleotides (ODNs) containing the fluorescent base analogue 2-aminopurine at the target positions. In addition, tighter binding of the * This work was supported by a grant from the Deutsche Forschungsgemeinschaft (We1453/3-2). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

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“…In this scenario, the stacking interaction between the DNA base and the Tyr836 may be beneficial for the geometry rather than energy barrier for the DNA base movement across the oscillating BH to determine the frequency of forward translocation. The energetic contribution of the RNA base-Tyr stacking interaction has been experimentally calculated as ∼2-5 k B T at room temperature (19,20), supporting this view.…”

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confidence: 60%

Unveiling translocation intermediates of RNA polymerase

Imashimizu

Kashlev

2014

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

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Translocation of RNA polymerase (RNAP) is a robust target for regulation of gene expression in prokaryotes and eukaryotes (1-3). During elongation, RNAP frequently encounters a broad range of DNA/RNA conformations (RNA hairpins, curved and cruciform DNA), DNA-binding proteins, DNA lesions, and misincorporation events at the 3′ ends of the RNA. These encounters impede forward translocation, leading to RNAP pausing (3). There are many protein factors that strengthen or weaken pausing by targeting translocation, such as archaeal/eukaryotic Spt5 and bacterial NusG/RfaH (4) or N/Nun proteins of lambdoid phages (3). In metazoa, RNAP II pausing in promoter-proximal regions plays a role in having polymerases in place for a rapid transcription response to environmental stimuli, such as heat-shock or cell differentiation, and maintaining a basal level of gene expression (5, 6). Translocation blocks also initiate RNAP backtracking (7). Backtracking events disengage the 3′ RNA end from the RNAP catalytic site, which stabilizes pausing; this type of event broadly controls gene transcription in eukaryotes (8). Forward translocation of RNAP along DNA has long been regarded as the movement of the RNA-DNA hybrid through the catalytic cleft, which vacates the active center, termed i+1 site, for an NTP binding. In this sense, the mechanism of translocation has been primarily focused on the hybrid movement, with only limited emphasis on the DNA sequences surrounding the hybrid (1, 2, 9). However, several reports indicate that the DNA sequences immediately upstream and downstream from the hybrid regulate translocation (7, 10-12). In PNAS, Silva et al. (13) identify an important component of the translocation mechanism using millisecond molecular dynamics (MD) simulation of translocation of yeast RNAP II: Translocation of the hybrid occurs before entry of the template DNA base to the i+1 site, where it can pair with an NTP. A similar scenario had been suggested based on the X-ray structure of RNAP II with the transcription inhibitor α-amanitin (9). The MD simulation posits that the entering of the template DNA base requires its stacking interaction with Tyr836 in the middle of the bridge helix (BH), a long α-helix separating the i+1 site from the downstream DNA (Fig. 1A). The Tyr residue in the BH is highly conserved from Escherichia coli to human. The bent and straight forms of the BH have been previously observed in the crystal structure of bacterial RNAP and eukaryotic RNAP II (14, 15), leading to one hypothesis that the bent-stretch transition or oscillation of the BH coupled with the movement of the trigger loop, another flexible part of the i+1 site involved in catalysis and substrate binding (16), is a driving force for forward translocation by forming a "pawl" (17). By revealing long-time translocation dynamics with atomic resolution, Silva et al. (13) show that the stacking interaction of the i+1 DNA base with the tyrosine of the BH may form an additional pawl.Based on kinetic modeling, Silva et al. argue that the template ...

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Mutagenesis of a stacking contact in the MS2 coat protein-RNA complex.

Cited by 59 publications

References 35 publications

Stacking interactions in PUF–RNA complexes

Stacking interactions in PUF–RNA complexes

Identification of the Binding Site for the Extrahelical Target Base in N 6-Adenine DNA Methyltransferases by Photo-cross-linking with Duplex Oligodeoxyribonucleotides Containing 5-Iodouracil at the Target Position

Unveiling translocation intermediates of RNA polymerase

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