Specific Interaction between RNA Phage Coat Proteins and RNA

Witherell, Gary W.; Gott, Jonatha M.; Uhlenbeck, Olke C.

doi:10.1016/s0079-6603(08)60842-9

Cited by 179 publications

(161 citation statements)

References 144 publications

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“…For two decades, this RNA element has been referred to as PS (70). The classical PS (i) is a single RNA hairpin (70,71) or a closely mapping cluster of structures (34), which bind strongly to a specific site in a CP; (ii) maps to a single genome locus that is distinct for related viruses (34); (iii) presents with a sequence that is conserved for a given virus species. Conservation of the classical PS allows the direct discovery of the PS by synplot2 computer analyses (32,33); (iv) triggers assembly in vitro or in vivo that can be prevented by site-directed mutagenesis of even single nucleotides (71); (v) can be transplanted into a different RNA bestowing to this heterologous RNA the ability to be encapsidated into the PS-cognate VLP (42); or (vi) can be transplanted into a different genome, thereby generating a chimeric virus (70).…”

Section: Discussionmentioning

confidence: 99%

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

Song¹,

Gorbatsevych²,

Liu³

et al. 2017

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

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Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2 Min , a variant temperature-sensitive at 33 and 39.5 • C. Compared with WT PV1, P2 Min displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2 Min , 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a role in determining packaging specificity do not survive codon pair recoding. This makes it unlikely that numerous "cryptic, poliovirus | genome recoding | packaging signal | specific infectivity T he capsid precursor P1 (881 amino acids) of type 1 poliovirus (PV), mapping to the N terminus of the polyprotein (PP) (Fig. 1A), can be encoded in 10 442 ways (1) due to the degenerate genetic code. The tiniest fraction of these possible sequences defines PV, the cause of poliomyelitis. PV occurs in three serotypes, of which the most neurovirulent type 1 Mahoney [PV1(M)], the main viral species analyzed in this study, was isolated in 1941 from pooled feces of three healthy children (2).Genome sequence (3, 4) and gene organization (3) of PV1(M) revealed highly complex structures in its 5'-terminal nontranslated region (5'-NTR), followed by a single ORF encoding the PP, followed by a complex 3'-heteropolymeric region and poly(A) tail (Fig. 1A) (5, 6). The PP (7) is an active molecule that cleaves itself into ∼29 polypeptides by two viral proteinases (2A pro and 3C pro /3CD pro ) and an enzyme-independent maturation cleavage (Fig. 1A) (5,6,8).Capsid domain P1 controls the identity of PV by determining virion structure (9), serotype identity (10), and interaction with the cellular receptor CD155 (10). Since PV replicates as quasispecies at an error rate of ∼10 −4 (11), the following questions arise: How conserved is its synonymous sequence given the astronomical number of alternative possibilities? What encoding could have coevolved that would be optimal to specify 881 capsid residues?If PV, a member of the genus Enterovirus of Picornaviridae, is an evolutionary descendant of C-cluster Coxsackie viruses (C-CAVs) (12), the evolution of PV nucleotide sequences was constrained as it adhered to the basic architecture of C-CAVs, its evolutionary parents (13). A second well-known restriction of sequence variability in ORFs is "codon bias" (14), the unequal use of synonymous codons. Encoding the PV1 P1 domain with an excess of "rarely used" (human) synonymous codons results in a ...

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Section: Discussionmentioning

confidence: 99%

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

Song¹,

Gorbatsevych²,

Liu³

et al. 2017

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

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“…The reason for selecting MS2 RNA- coat protein crosslinking as a model is that this system has been characterized by a cocrystal structure as well as with photocrosslinking agents (Witherell et al 1991;Valegard et al 1994;Stockley et al 1995). Different concentrations (0-200 ng) of E. coli-expressed MS2 coat protein were incubated with 32 P-labeled hairpin RNA (∼10 fmole), and the reaction mixture was irradiated with long-wavelength UV light with a hand-held UV source.…”

Section: Rna-protein Crosslinking With 8-azidoadenosine-containing Rnamentioning

confidence: 99%

Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase

Gopalakrishna¹,

Gusti²,

Nair³

et al. 2004

RNA

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UV-induced photochemical crosslinking is a powerful approach that can be used for the identification of specific interactions involving nucleic acid-protein and nucleic acid-nucleic acid complexes. 8-AzidoATP (8-N 3 ATP) is a photoaffinity-labeling agent which has been widely used to elucidate the ATP binding site of a variety of proteins. However, its true potential as a photoactivatable nucleotide analog could not be exploited due to the lack of 8-azidoadenosine phosphoramidite, a monomer used in the synthesis of RNA, and the inability of 8-N 3 ATP to serve as an efficient substrate for bacteriophage RNA polymerase. In this study, we explored the ability of SP6, T3, and T7 RNA polymerases and metal ion cofactors to catalyze the incorporation of 8-N 3 AMP into RNA. Whereas transcription buffer containing 2.0-2.5 mM Mn 2+ supports T7 RNA polymerase-mediated insertion of 8-N 3 AMP into RNA, a mixture of 2.5 mM Mn 2+ and 2.5 mM Mg 2+ further improves the yield of 8-N 3 AMP-containing transcript. In addition, both RNA transcription and reverse transcription proceed with high fidelity for the incorporation of 8-N 3 AMP and complementary residue, respectively. Finally, we show that a high-affinity MS2 coat protein binding sequence, in which adenosine residues were replaced by 8-azidoadenosine, crosslinks to the coat protein of the Escherichia coli phage MS2.

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“…A chimeric protein consisting of MS2 bacteriophage coat protein linked to yeast Pab1p (MS2/Pab1p) was expressed in yeast; the same strain also carried a second plasmid expressing a reporter mRNA with MS2 recognition sites in its 3Ј UTR. Two recognition sites were inserted, as binding of MS2 coat protein to adjacent sites is cooperative (Witherell et al 1991). This approach separated analysis of the function of Pab1p from its ability to bind poly(A) or support cell growth.…”

Section: Experimental Strategymentioning

confidence: 99%

“…These results suggest that tethered Pab1p stabilizes an mRNA that has had its tail largely, or completely, removed. Mutant sites bind in vitro with an 100-fold reduction in affinity (Witherell et al 1991). Black dot is at left of each group of three lanes indicates positions of specific complexes.…”

Section: Tethered Pab1p Stabilizes Partially and Fully Deadenylated Mmentioning

confidence: 99%

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation

1998

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Translation and mRNA stability are enhanced by the presence of a poly(A) tail. In vivo, the tail interacts with a conserved polypeptide, poly(A) binding protein (Pab1p). To examine Pab1p function in vivo, we have tethered Pab1p to the 3 UTR of reporter mRNAs by fusing it to MS2 coat protein and placing MS2 binding sites in the 3 UTR of the reporter. This strategy allows us to uncouple Pab1p function from its RNA binding activity. We show that mRNAs that lack a poly(A) tail in vivo are stabilized by Pab1p, and that the portions of Pab1p required for stabilization are genetically distinct from those required for poly(A) binding. In addition, stabilization by Pab1p requires ongoing translation of the mRNA. We conclude that the primary, or sole, function of poly(A) with respect to mRNA stability is simply to bring Pab1p to the mRNA, and that mRNA stabilization is an intrinsic property of Pab1p. The approach we describe may be useful in identifying and assaying 3 UTR regulatory proteins, as it uncouples analysis of function from RNA binding.

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Specific Interaction between RNA Phage Coat Proteins and RNA

Cited by 179 publications

References 144 publications

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation

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Specific Interaction between RNA Phage Coat Proteins and RNA

Cited by 179 publications

References 144 publications

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

Template-dependent incorporation of 8-N3AMP into RNA with bacteriophage T7 RNA polymerase

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation

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Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase