On the nature of spontaneous RNA synthesis by Qβ replicase

Chetverin, Alexander B.; Chetverina, Helena V.; Munishkin, Alexander

doi:10.1016/0022-2836(91)90729-p

Cited by 65 publications

(59 citation statements)

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“…The used variant of RQ135 RNA was prepared by transcription of SmaI-digested plasmid pT7RQ135 -1 (-) 42 modified by inserting CGAUCC between positions 52 and 53 of the original RQ135 -1 (-) sequence; the template properties of this variant were indistinguishable 35 from those of the authentic RQ135 RNA 22 . Qb( þ ) RNA was isolated from purified wild-type phage particles lysed with SDS, followed by centrifugation through a 15% sucrose cushion and phenol extraction 25 . Qb( À ) RNA was prepared by transcription of SmaI-digested plasmid pQb8 30 .…”

Section: Methodsmentioning

confidence: 99%

“…The concept that S1 is exclusively needed for initiation on the Qb( þ ) RNA and is not needed for initiation on other templates was based on data obtained in a high-salt buffer 7 . The authors argued that salt helped to avoid the interfering 'spontaneous' RNA synthesis that was later shown to be caused by traces of contaminating RQ RNAs 25 . Further, the authors of the concept measured the total RNA synthesis rather than the initiation resulting in the full-sized template-specific products; they assumed that S1 promoted the synthesis through enhanced initiation, but neither they nor others ever tested this hypothesis directly.…”

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confidence: 99%

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Ribosomal protein S1 functions as a termination factor in RNA synthesis by Qβ phage replicase

et al. 2013

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S1 is the largest ribosomal protein, and is vitally important for the cell. S1 is also a subunit of Qb replicase, the RNA-directed RNA polymerase of bacteriophage Qb. In both protein and RNA syntheses, S1 is commonly believed to bind to a template RNA at the initiation step, and not to be involved in later events. Here, we show that in Qb replicase-mediated RNA synthesis, S1 functions at the termination step by promoting release of the product strand in a single-stranded form. This function is fulfilled by the N-terminal fragment comprising the first two S1 domains. The results suggest that S1 might also have a role other than mRNA binding in the ribosome.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Ribosomal protein S1 functions as a termination factor in RNA synthesis by Qβ phage replicase

et al. 2013

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“…However, there are relatively few RSV-specific sequences in the minigenome construct, and it would be expected that encapsidation of the template would obscure any internal initiation site. A third possibility is that the RSV polymerase might have the capacity to use any RNA as a template, similarly to the polymerase of phage Qb (Hill and Blumenthal 1983;Chetverin et al 1991;Ugarov et al 2003), allowing it to initiate abortive RNA synthesis at a UG sequence to create a dinucleotide primer that it can then use to initiate RNA replication at the 39 end of the genome. A fourth possibility is that the RSV polymerase is able to recruit oligonucleotides from the cytoplasmic milieu to act as primers.…”

Section: ''Template-independent'' Initiationmentioning

confidence: 99%

The first two nucleotides of the respiratory syncytial virus antigenome RNA replication product can be selected independently of the promoter terminus

Noton

Fearns²

2011

RNA

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There is limited knowledge regarding how the RNA-dependent RNA polymerases of the nonsegmented negative-strand RNA viruses initiate genome replication. In a previous study of respiratory syncytial virus (RSV) RNA replication, we found evidence that the polymerase could select the 59-ATP residue of the genome RNA independently of the 39 nucleotide of the template. To investigate if a similar mechanism is used during antigenome synthesis, a study of initiation from the RSV leader (Le) promoter was performed using an intracellular minigenome assay in which RNA replication was restricted to a single step, so that the products examined were derived only from input mutant templates. Templates in which Le nucleotides 1U, or 1U and 2G, were deleted directed efficient replication, and in both cases, the replication products were initiated at the wild-type position, at position -1 or -2 relative to the template, respectively. Sequence analysis of the RNA products showed that they contained ATP and CTP at the -1 and -2 positions, respectively, thus restoring the mini-antigenome RNA to wild-type sequence. These data indicate that the RSV polymerase is able to select the first two nucleotides of the antigenome and initiate at the correct position, even if the 39-terminal two nucleotides of the template are missing. Substitution of positions +1 and +2 of the template reduced RNA replication and resulted in increased initiation at positions +3 and +5. Together these data suggest a model for how the RSV polymerase initiates antigenome synthesis.

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“…the RNA is only present at low levels as a contaminant, and that, whilst the Qb replicase can initiate RNA synthesis randomly, it does not enter a stable elongation mode if initiation occurs in the absence of the appropriate promoter (Chetverin et al, 1991;Hill & Blumenthal, 1983;Ugarov et al, 2003). Thus, an explanation for the TCV-mediated repair is that, in the absence of an appropriate promoter sequence, the viral replicase generates RNA in a template-independent manner or initiates randomly on any RNA available, thus generating a pool of random oligonucleotides, some of which could fit appropriately with the RdRp to be utilized as primers to initiate replication, similarly to the mechanism described above.…”

Section: Non-templated Polymerization By the Viral Replicase To Genermentioning

confidence: 99%

How RNA viruses maintain their genome integrity

Barr

Fearns

2010

Journal of General Virology

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On the nature of spontaneous RNA synthesis by Qβ replicase

Cited by 65 publications

References 27 publications

Ribosomal protein S1 functions as a termination factor in RNA synthesis by Qβ phage replicase

Ribosomal protein S1 functions as a termination factor in RNA synthesis by Qβ phage replicase

The first two nucleotides of the respiratory syncytial virus antigenome RNA replication product can be selected independently of the promoter terminus

How RNA viruses maintain their genome integrity

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