Altering the Context of an RNA Bulge Switches the Binding Specificities of Two Viral Tat Proteins

Smith, Colin A.; Crotty, Shane; Harada, Yuka; Frankel, Alan D.

doi:10.1021/bi980382+

Cited by 30 publications

(53 citation statements)

References 33 publications

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“…Interestingly, one of these mutations, the insertion of an adenosine at position 24 of the ribozyme, is similar to a modification previously identified in the wild-type TAR site that increases the affinity of the site Ϸ3-fold (33). The higher affinity was attributed to the creation of a less constrained TAR conformation or increased major groove accessibility (33). Other mutations clustered in the central region of the molecule proximal to stem 3.…”

Section: Resultsmentioning

confidence: 99%

A ribozyme that ligates RNA to protein

Baskerville

Bartel

2002

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

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We have used a combination of in vitro selection and rational design to generate ribozymes that form a stable phosphoamide bond between the 5 terminus of an RNA and a specific polypeptide. This reaction differs from that of previously identified ribozymes, although the product is analogous to the enzymenucleotidyl intermediates isolated during the reactions of certain proteinaceous enzymes, such as guanyltransferase, DNA ligase, and RNA ligase. Comparative sequence analysis of the isolated ribozymes revealed that they share a compact secondary structure containing six stems arranged in a four-helix junction and branched pseudoknot. An optimized version of the ribozyme reacts with substrate-fusion proteins, allowing it to be used to attach RNA tags to proteins both in vitro and within bacterial cells, suggesting a simple way to tag a specific protein with amplifiable information.in vitro selection ͉ nucleic acid tag ͉ phosphoamide ͉ Tat ͉ TAR I n vitro selection has been used in combination with rational design to identify and optimize numerous ribozymes and binding motifs (1-3). The results of these experiments continue to give insight into the capacity of RNA and DNA to catalyze a variety of chemical reactions and the ability of nucleic acids to recognize an array of substrates. Some of these catalysts are being developed into effective tools for use in molecular biology. These include allosteric ribozymes, used as detectors of small molecules and proteins, and DNA enzymes that efficiently cleave target sequences (4-6).Here we use in vitro selection coupled with rational design to produce a new ribozyme that ligates RNA to protein through the formation of a phosphoamide bond. This is similar to a reaction found in naturally occurring proteins such as guanyltransferase, and DNA and RNA ligases that form phosphoamide bonds as enzyme-nucleotidyl intermediates during cap formation and ligation events (7-9). The selection of this new ribozyme expands the repertoire of RNA-catalyzed chemistry and further illustrates the ability of RNA to chemically modify a variety of substrates. Moreover, this ribozyme might be used to synthesize oligonucleotide-tagged proteins in vitro and in vivo. These attached oligonucleotides might be useful as affinity tags or could function as amplifiable identifying markers. For example, this ribozyme might be used to attach mRNAs to the proteins that they encode. A diverse library of mRNA-protein fusions could be used in conjunction with in vitro selection strategies to identify previously undescribed proteins with unique or enhanced properties, as has been shown previously by using phage display, ribosome display, and mRNA display (10-13). Materials and MethodsPool Construction. The double-stranded DNA pool was constructed from two 110-nt DNA fragments, each containing 76 completely randomized positions (14). Each fragment was amplified separately in 110 ml PCR reactions and ligated through BanI sites to generate a final pool with the sequence: TTCTAATACGACTCACTATAG-GACAGCTCCGAGCATTCTCGTGT...

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Section: Resultsmentioning

confidence: 99%

A ribozyme that ligates RNA to protein

Baskerville

Bartel

2002

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

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“…Other examples are known where base pairs influence the stability of proximal loops, such as that of boxB (24) and a related tetraloop (46). Interestingly, U:A and A:U base pairs adjacent to the loop of bovine immunodeficiency virus (BIV) trans-acting responsive element indirectly modulate the binding of the BIV Tat peptide to the stem (41). We speculate that the cytosines of P22 boxB right are tolerated because they are paired and stacked between the loop and the stem, as proposed for the homologous C:C base pair in phage 21 boxB (7).…”

Section: Discussionmentioning

confidence: 99%

“…NMR studies reveal that HIV RRE binds to its cognate peptide HIV Rev and to a selected peptide, RSG1.2, maintaining the same base pairing (50). A mutant hairpin RNA is able to function in either HIV or BIV transcriptional transactivation by binding homologous argininerich peptides in different recognition modes (41 Relaxed specificity may require the ability to adopt distinct conformations. Though plasticity comes with lowered thermodynamic stability, evolution presents many examples of conformational flexibility.…”

Section: Discussionmentioning

confidence: 99%

“…Though computational modeling of evolving RNA secondary structure strongly supports neutral theories (21,38,45), few experimental tests using biologically active RNAs have been reported. Studies of small hairpin RNAs that bind arginine-rich peptides have shown that single substitutions and base pair changes are enough to create changes of specificity (23,41), suggesting that neutral paths between distinct activities can be found when sequences are small. In the case of protein-binding RNAs, mutations leading from relaxed-specificity sequences to sequences with distinct binding preferences would be equivalent to speciation.…”

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Bacteriophage P22 Antitermination boxB Sequence Requirements Are Complex and Overlap with Those of λ

2008

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Transcription antitermination in phages and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. A nuclear magnetic resonance structure of the P22 N peptide boxB left complex and limited mutagenesis have been reported but do not reveal a consensus sequence for boxB. We have used a plasmidbased antitermination system to screen boxBs with random loops and to test boxB mutants. We find that P22 N requires boxB to have a GNRA-like loop with no simple requirements on the remaining sequences in the loop or stem. U:A or A:U base pairs are strongly preferred adjacent to the loop and appear to modulate N binding in cooperation with the loop and distal stem. A few GNRA-like hexaloops have moderate activity. Some boxB mutants bind P22 and N, indicating that the requirements imposed on boxB by P22 N overlap those imposed by N. Point mutations can dramatically alter boxB specificity between P22 and N. A boxB specific for P22 N can be mutated to N specificity by a series of single mutations via a bifunctional intermediate, as predicted by neutral theories of evolution.and P22 share a closely related system of regulating the expression of early lytic genes by allowing transcription past terminators in the P left and P right operons (47). The recognition of the boxB RNA hairpins of nut (N-utilization) sites by the binding domains of the viral N proteins (Fig. 1A, B, and C) initiates the assembly of an antitermination complex. This complex contains N, host factor NusA, RNA polymerase, and other host factors bound to the viral nut site boxB and the nonhairpin boxA, allowing transcription to proceed through downstream transcription termination signals (32). The four wild-type nut sites of and P22 are similar in sequence but differ even between boxB left and boxB right in the same virus. Notably, both P22 boxBs have a C in the loop where both boxBs have an A, P22 boxB stems are longer than those of by 1 base pair, and P22 boxB right appears to have a noncanonical C:C base pair. boxBs bind noncognate N peptides poorly in vitro (2,8,44). Likewise, noncognate N-nut interactions do not function in vivo (14, 28), and noncognate N proteins do not rescue N-deficient viruses (10).The details of the N-boxB interaction have been revealed by extensive genetic and biochemical work (47) and by solution state nuclear magnetic resonance (NMR) structures of the arginine-rich domain of the N protein bound to boxB left (29) and boxB right (39) RNA. Genetic and biochemical studies of the P22 interaction are less complete (13, 44) but are supported by the solution state NMR structure of the arginine-rich domain of the N protein bound to P22 boxB right (5).and P22 boxBs bind their N peptides as hairpins in which 4 of 5 bases adopt a GNRA-like tetraloop structure (Fig. 2). Tetraloops are frequently found in RNAs serving structural roles as stable caps to stems and as motifs recognized by proteins; GNRA tetraloops are noted ...

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“…1B), and their bound three-dimensional conformations around the bulge regions are very similar (Fig. 1C) (30). Even with these similarities, the BIV Tat ARM cannot adopt the high-affinity ␤-hairpin conformation with HIV TAR.…”

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confidence: 89%

A single intermolecular contact mediates intramolecular stabilization of both RNA and protein

Calabro

Daugherty

Frankel

2005

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

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An arginine-rich peptide from the Jembrana disease virus (JDV) Tat protein is a structural ''chameleon'' that binds bovine immunodeficiency virus (BIV) or HIV TAR RNAs in two different binding modes, with an affinity for BIV TAR even higher than the cognate BIV peptide. We determined the NMR structure of the JDV Tat-BIV TAR high-affinity complex and found that the C-terminal tyrosine in JDV Tat forms a network of inter-and intramolecular hydrogen bonding and stacking interactions that simultaneously stabilize the ␤-hairpin conformation of the peptide and a base triple in the RNA. A neighboring histidine also appears to help stabilize the peptide conformation. Induced fit binding is recurrent in protein-protein and protein-nucleic acid interactions, and the JDV Tat complex demonstrates how high affinity can be achieved not only by optimization of the binding interface but also by inducing new intramolecular contacts that stabilize each binding partner. Comparison to the cognate BIV Tat peptide-TAR complex shows how such a costabilization mechanism can evolve with only small changes to the peptide sequence. In addition, the bound structure of BIV TAR in the chameleon peptide complex is strikingly similar to the bound conformation of HIV TAR, suggesting new strategies for the development of HIV TAR binding molecules.NMR ͉ RNA structure ͉ RNA-binding domain T he ability of macromolecules to interact with high affinity and specificity is often accompanied by conformational changes in the binding partners (1-4). Flexibility of one or both molecules can contribute to optimization of the binding surfaces and allow binding to multiple partners (5, 6). Numerous examples of conformational adaptability have been observed in protein-protein and protein-nucleic acid interactions, including Ig proteins, which can accommodate a remarkably wide range of binding partners, and the ribosome, where induced-fit binding helps direct its ordered assembly (7-9).In RNA-protein interactions, the arginine-rich motif (ARM) has served as a model system for examining structural mechanisms that underlie induced fit binding (10-12). Studies of ARM peptide-RNA complexes, including the Tat ARM-TAR RNA interactions of several lentiviruses, have shown that the unbound ARMs generally are unfolded and can adopt a variety of conformations upon RNA binding, often with a concomitant change in RNA structure. In the case of HIV Tat, a relatively weak binding ARM peptide remains in an extended conformational when bound to HIV TAR but causes a large conformational change in the RNA, inducing stacking between the two helical stems and formation of a U-A:U base triple (13-23). In the case of bovine immunodeficiency virus (BIV) Tat, a conformational change in BIV TAR also is observed upon binding, but the cognate ARM peptide undergoes a large conformational rearrangement, forming a ␤-hairpin structure that facilitates high-affinity binding through a large set of specific contacts to the RNA (24-29). Despite the striking difference in binding modes of these two...

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Altering the Context of an RNA Bulge Switches the Binding Specificities of Two Viral Tat Proteins

Cited by 30 publications

References 33 publications

A ribozyme that ligates RNA to protein

A ribozyme that ligates RNA to protein

Bacteriophage P22 Antitermination boxB Sequence Requirements Are Complex and Overlap with Those of λ

A single intermolecular contact mediates intramolecular stabilization of both RNA and protein

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