A complete plasmid-based complementation system for RNA coliphage Qβ: Three proteins of bacteriophages Qβ (Group III) and SP (Group IV) can be interchanged

Priano, Christine; Arora, Ruchi; Butke, John; Mills, Donald R.

doi:10.1006/jmbi.1995.0297

Cited by 17 publications

(20 citation statements)

References 25 publications

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“…Induction of E. coli BL21(DE3) host transformants with IPTG results in transcription of the entire 4217 nucleotide Qb RNA plus-strand sequence. When wild-type Qb sequences are produced, an infectious cycle ensues during which mature Qb phage particles are generated and released upon lysis of the host (Mills et al, 1989(Mills et al, , 1990Priano et al, 1995;Arora et al, 1996).…”

Section: Resultsmentioning

confidence: 98%

Translational activation in coliphage qβ: on a polycistronic messenger RNA, repression of one gene can activate translation of another 1 1Edited by M. Gottesman

Priano

Arora

Jayant

et al. 1997

Journal of Molecular Biology

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Section: Resultsmentioning

confidence: 98%

Translational activation in coliphage qβ: on a polycistronic messenger RNA, repression of one gene can activate translation of another 1 1Edited by M. Gottesman

Priano

Arora

Jayant

et al. 1997

Journal of Molecular Biology

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“…Selection against RT − mutants was ameliorated by using a complementing system in trans based on a plasmid that encodes the entire Coat/RT mRNA with the natural UGA stop codon replaced with a TGG tryptophan codon [35]. To further reduce the effect of selection, the experimental Qß populations were established after a single cycle of infection.…”

Section: Introductionmentioning

confidence: 99%

The Three Faces of Riboviral Spontaneous Mutation: Spectrum, Mode of Genome Replication, and Mutation Rate

Garcı́a-Villada¹,

Drake

2012

PLoS Genet

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Riboviruses (RNA viruses without DNA replication intermediates) are the most abundant pathogens infecting animals and plants. Only a few riboviral infections can be controlled with antiviral drugs, mainly because of the rapid appearance of resistance mutations. Little reliable information is available concerning i) kinds and relative frequencies of mutations (the mutational spectrum), ii) mode of genome replication and mutation accumulation, and iii) rates of spontaneous mutation. To illuminate these issues, we developed a model in vivo system based on phage Qß infecting its natural host, Escherichia coli . The Qß RT gene encoding the Read-Through protein was used as a mutation reporter. To reduce uncertainties in mutation frequencies due to selection, the experimental Qß populations were established after a single cycle of infection and selection against RT − mutants during phage growth was ameliorated by plasmid-based RT complementation in trans . The dynamics of Qß genome replication were confirmed to reflect the linear process of iterative copying (the stamping-machine mode). A total of 32 RT mutants were detected among 7,517 Qß isolates. Sequencing analysis of 45 RT mutations revealed a spectrum dominated by 39 transitions, plus 4 transversions and 2 indels. A clear template•primer mismatch bias was observed: A•C>C•A>U•G>G•U> transversion mismatches. The average mutation rate per base replication was ≈9.1×10 −6 for base substitutions and ≈2.3×10 −7 for indels. The estimated mutation rate per genome replication, μ g , was ≈0.04 (or, per phage generation, ≈0.08), although secondary RT mutations arose during the growth of some RT mutants at a rate about 7-fold higher, signaling the possible impact of transitory bouts of hypermutation. These results are contrasted with those previously reported for other riboviruses to depict the current state of the art in riboviral mutagenesis.

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“…The plasmid pT7QβMat(+) is a variation of pT7Qβ(+)500 with nucleotides 1406–4217 of the Qβ cDNA sequence deleted (23,24). …”

Section: Methodsmentioning

confidence: 99%

In polycistronic Q RNA, single-strandedness at one ribosome binding site directly affects translational initiations at a distal upstream cistron

Jayant¹,

Priano²,

Mills³

2010

Nucleic Acids Research

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In Qβ RNA, sequestering the coat gene ribosome binding site in a putatively strong hairpin stem structure eliminated synthesis of coat protein and activated protein synthesis from the much weaker maturation gene initiation site, located 1300 nucleotides upstream. As the stability of a hairpin stem comprising the coat gene Shine–Dalgarno site was incrementally increased, there was a corresponding increase in translation of maturation protein. The effect of the downstream coat gene ribosome binding sequence on maturation gene expression appeared to have occurred only in cis and did not require an AUG start codon or initiation of coat protein synthesis. In all cases, no structural reorganization was predicted to occur within Qβ RNA. Our results suggest that protein synthesis from a relatively weak translational initiation site is greatly influenced by the presence or absence of a stronger ribosome binding site located elsewhere on the same RNA molecule. The data are consistent with a mechanism in which multiple ribosome binding sites compete in cis for translational initiations as a means of regulating protein synthesis on a polycistronic messenger RNA.

show abstract

A complete plasmid-based complementation system for RNA coliphage Qβ: Three proteins of bacteriophages Qβ (Group III) and SP (Group IV) can be interchanged

Cited by 17 publications

References 25 publications

Translational activation in coliphage qβ: on a polycistronic messenger RNA, repression of one gene can activate translation of another 1 1Edited by M. Gottesman

Translational activation in coliphage qβ: on a polycistronic messenger RNA, repression of one gene can activate translation of another 1 1Edited by M. Gottesman

The Three Faces of Riboviral Spontaneous Mutation: Spectrum, Mode of Genome Replication, and Mutation Rate

In polycistronic Q RNA, single-strandedness at one ribosome binding site directly affects translational initiations at a distal upstream cistron

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