Evolvability of the mode of peptide binding by an RNA

Iwazaki, Tetsuya; Li, Xianglan; Harada, Kaoru

doi:10.1261/rna.2560905

Cited by 23 publications

(35 citation statements)

References 40 publications

(87 reference statements)

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“…However, experimental evidence with biologically active RNAs has been limited. Experimental support for the application of neutral theories to RNA activity has come from only a few examples (18,22,23,40). Our findings that single substitutions connect a P22 N-specific boxB via a moderately bifunctional boxB to a N-specific boxB support the extension of neutral theories from computationally predictable RNA secondary structures to biologically active RNA phenotypes.…”

Section: Discussionmentioning

confidence: 53%

“…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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confidence: 99%

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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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Section: Discussionmentioning

confidence: 53%

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confidence: 99%

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

2008

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“…The reciprocal mechanism of adopting both P22 and boxB loop conformations appears to be employed by relaxed-specificity boxBs (11). Relaxedspecificity sequences may be able to acquire specificity simply by mutations that discriminate against one target without affecting binding to another, as has been seen in HIV RRE (26).…”

Section: Discussionmentioning

confidence: 99%

The RNA-Binding Domain of Bacteriophage P22 N Protein Is Highly Mutable, and a Single Mutation Relaxes Specificity toward λ

2008

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Antitermination in bacteriophage P22, a lambdoid phage, uses the arginine-rich domain of the N protein to recognize boxB RNAs in the nut site of two regulated transcripts. Using an antitermination reporter system, we screened libraries in which each nonconserved residue in the RNA-binding domain of P22 N was randomized. Mutants were assayed for the ability to complement N-deficient virus and for antitermination with P22 boxB left and boxB right reporters. Single amino acid substitutions complementing P22 N ؊ virus were found at 12 of the 13 positions examined. We found evidence for defined structural roles for seven nonconserved residues, which was generally compatible with the nuclear magnetic resonance model. Interestingly, a histidine can be replaced by any other aromatic residue, although no planar partner is obvious. Few single substitutions showed bias between boxB left and boxB right , suggesting that the two RNAs impose similar constraints on genetic drift. A separate library comprising only hybrids of the RNA-binding domains of P22, , and 21 N proteins produced mutants that displayed bias. P22 N ؊ plaque size plotted against boxB left and boxB right reporter activities suggests that lytic viral fitness depends on balanced antitermination. A few N proteins were able to complement both N-and P22 N-deficient viruses, but no proteins were found to complement both P22 N-and 21 N-deficient viruses. A single tryptophan substitution allowed P22 N to complement both P22 and N ؊ . The existence of relaxed-specificity mutants suggests that conformational plasticity provides evolutionary transitions between distinct modes of RNA-protein recognition.Lambdoid phages , P22, 21, and other phages regulate the expression of delayed early genes by allowing transcription past terminators in the P left and P right operons. These phages share regulatory mechanisms with , but they have uncertain evolutionary relationships that are obscured by recombination among tailed phages (Caudovirales) (7,8). In phage , antitermination allows expression of genes regulating the development of lysis or lysogeny (12). The assembly of transcription antitermination complexes in P22, , and 21 is initiated by the binding of viral N proteins to small hairpin boxB RNAs in the nut sites (N utilization) of regulated transcripts (40). These complexes contain N and host factors, including NusA and the transcribing polymerase, allowing transcription to proceed through downstream transcription termination signals. P22, , and 21 exhibit type specificity, where the N protein of one virus cannot complement its absence in a different virus (13,24). N proteins recognize their cognate boxB RNAs via argininerich domains near their amino termini (21). Their boxBs are hairpin RNAs that have little sequence similarity, yet similar secondary structures (Fig. 1A). Alignment of P22, , and 21 reveals that N protein RNA-binding domains contain four conserved amino acids (Fig. 1B). Protein and RNA sequence differences create specificity; noncognate interactions functi...

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“…Despite a decrease in anti-termination activity upon introduction of heterologous interactions, presumably due to the loss of the interaction between the NusA protein and the boxB/N complex, the two-plasmid system has been shown to be a sensitive method for the analysis of small changes in binding affinity of RNA-polypeptide interactions. This bacterial system has also been shown to be a powerful tool in the screening and selection of novel RNA-binding peptides targeting the HIV Revresponse element (RRE) from combinatorial libraries (Harada et al, 1996;Peled-Zehavi et al, 2003;Sugaya et al, 2008a), as well as for the selection of novel peptide-binding RNAs (Iwazaki et al, 2005;Sugaya et al, 2008b). However, there appeared to be limitations on the size of RNAs that can be accommodated in the antitermination complex, with only the interaction of relatively small RNA stem-loops with their cognate polypeptides being detected using the two-plasmid system.…”

Section: Introductionmentioning

confidence: 99%

Replacement of the λ boxB RNA–N peptide with heterologous RNA–peptide interactions relaxes the strict spatial requirements for the formation of a transcription anti‐termination complex

Horiya¹,

Inaba²,

Koh³

et al. 2009

Molecular Microbiology

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SummaryIn bacteriophage l, formation of a transcriptional anti-termination complex involving the elongating RNA polymerase is mediated by the interaction of boxB RNA with the RNA-binding domain of the N protein (N peptide). In an attempt to understand the spatial requirements for boxB/N peptide interaction within the anti-termination complex, the effects of changes in the distance between boxA and boxB RNA, the length of the boxB stem, and the distance between the N peptide and remainder of the N protein were examined using a bacterial reporter system. It was found that the requirements for boxB stem length and the distance between N peptide and the remainder of N were optimized and strict. In contrast, replacement of the boxB/N interaction by heterologous RNA-peptide interactions appeared to relax the strict requirement for RNA stem length and the orientation of the RNA-binding peptide, presumably due to the absence of the cooperative interaction between boxB/N and the host factor NusA. In addition, the decrease in activity upon stem lengthening could be partially suppressed by simultaneous lengthening of the RNA spacer. A further understanding of the structural organization of the anti-termination complex may provide insights into how functional ribonucleoprotein complexes may be engineered.

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Evolvability of the mode of peptide binding by an RNA

Cited by 23 publications

References 40 publications

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

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

The RNA-Binding Domain of Bacteriophage P22 N Protein Is Highly Mutable, and a Single Mutation Relaxes Specificity toward λ

Replacement of the λ boxB RNA–N peptide with heterologous RNA–peptide interactions relaxes the strict spatial requirements for the formation of a transcription anti‐termination complex

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