Design of Artificial Short-Chained RNA Species That Are Replicated by Q.beta. Replicase

Zamora, Humberto; Luce, Ruediger; Biebricher, Christof Κ.

doi:10.1021/bi00004a020

Cited by 39 publications

(56 citation statements)

References 30 publications

(48 reference statements)

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“…A single-phage burst releases some 10,000 descendants per cell, of which only 5-10% appear infectious (1). A large number of the progeny phage carry single substitutions, but multiple base changes and recombinants are present as well (2)(3)(4). This collection of mutant genomes, the so-called quasispecies, allows the phage population to rapidly adapt to changes in its environment (5,6).…”

mentioning

confidence: 99%

Evolutionary reconstruction of a hairpin deleted from the genome of an RNA virus.

Olsthoorn

Duin

1996

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

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The intercistronic region between the maturation and coat-protein genes of RNA phage MS2 contains important regulatory and structural information. The sequence participates in two adjacent stem-loop structures, one of which, the coat-initiator hairpin, controls coat-gene translation and is thus under strong selection pressure. We have removed 19 out of the 23 nucleotides constituting the intercistronic region, thereby destroying the capacity of the phage to build the two hairpins. The deletion lowered coat-protein yield more than 1000-fold, and the titer of the infectious clone carrying the deletion dropped 10 orders of magnitude as compared with the wild type. Two types of revertants were recovered. One had, in two steps, recruited 18 new nucleotides that served to rebuild the two hairpins and the lost ShineDalgarno sequence. The other type had deleted an additional six nucleotides, which allowed the reconstruction of the ShineDalgarno sequence and the initiator hairpin, albeit by sacrificing the remnants of the other stem-loop. The results visualize the immense genetic repertoire created by, what appears as, random RNA recombination. It would seem that in this genetic ensemble every possible new RNA combination is represented.The single-stranded RNA coliphages are of mRNA polarity and are about 4000-nucleotides long. The genome encodes four proteins, needed for RNA replication and for the construction and spreading of the virions (Fig. 1A). New plus strands are generated via a minus-strand intermediate by an error-prone replicase. A single-phage burst releases some 10,000 descendants per cell, of which only 5-10% appear infectious (1). A large number of the progeny phage carry single substitutions, but multiple base changes and recombinants are present as well (2-4). This collection of mutant genomes, the so-called quasispecies, allows the phage population to rapidly adapt to changes in its environment (5, 6).Besides encoding the phage proteins the nucleotide sequence endows the RNA with the proper phenotype, i.e., it prescribes the proper secondary structure needed for translational control mechanisms, protein binding sites and other functions (7).To understand the significance of this phenotype we have constructed an infectious MS2 cDNA clone and used this clone to introduce changes that compromise preselected structure elements.One particularly delicate structure element is located around the start of the major coat protein (CP) gene. For group I phage MS2 this element is shown in Fig. lB. The sequence folds into two hairpins, one of which has the CP start codon in the loop, while the lower part of the stem contains the Shine-Dalgarno (SD) sequence (boxed), which mediates ribosome binding. In a previous evolutionary experiment we could show that the thermodynamic stability of this hairpin is of great

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confidence: 99%

Evolutionary reconstruction of a hairpin deleted from the genome of an RNA virus.

Olsthoorn

Duin

1996

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

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“…Most natural switches are either too large (more than 300 nt in length) or to complex (contain pseudoknot motifs) to be of interest for our purpose. Aside from the two SV11 molecules Biebricher and Luce (1992); Zamora et al (1995) we have included a number of nucleic acid logic gates that have been engineered in the laboratory as part of the dataset for the comparison study. Although the selection of nucleic acid logic gates may seem inappropriate for self-induced structural switching because the logic gates follow the trans-acting switches, we use them because these molecules are known to have multi-stable conformations.…”

Section: Evaluating the Performance Of Multi-stable Sequence Designersmentioning

confidence: 99%

“…RNA molecules with this switching mechanism are also known as trans-acting switches Nagel and Pleij (2002). In the laboratory, the conformational switching can also be achieved through the process of melting and rapid quenching of the molecule, illustrated by the multi-stable SV11 molecule Biebricher and Luce (1992); Zamora et al (1995) (Biebricher and Luce, 1992). The molecule has predominantly two confirmations.…”

Section: Introductionmentioning

confidence: 99%

Design of interacting multi-stable nucleic acids for molecular information processing

Ramlan

Zauner

2011

Biosystems

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Abstract. Despite an exponential increase in computing power over the past decades, present information technology falls far short of expectations in areas such as cognitive systems and micro robotics. Organisms demonstrate that it is possible to implement information processing in a radically different way from what we have available in present technology, and that there are clear advantages from the perspective of power consumption, integration density, and real-time processing of ambiguous data. Accordingly, the question whether the current silicon substrate and associated computing paradigm is the most suitable approach to all types of computation has come to the fore. Macromolecular materials, so successfully employed by nature, possess uniquely promising properties as an alternate substrate for information processing. The two key features of macromolecules are their conformational dynamics and their self-assembly capabilities. The purposeful design of macromolecules capable of exploiting these features has proven to be a challenge, however, for some groups of molecules it is increasingly practicable. We here introduce an algorithm capable of designing groups self-assembling of nucleic acid molecules with multiple conformational states. Evaluation using natural and artificially designed nucleic acid molecules favours this algorithm significantly, as compared to the probabilistic approach. Furthermore, the thermodynamic properties of the generated candidates are within the same approximation as the customised trans-acting switching molecules reported in the laboratory.

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“…Unlike the RdRp of any eukaryotic positive-strand RNA virus, Q␤ replicase has been purified to homogeneity and shown to be capable of supporting the full viral genome amplification cycle in vitro (6). This enzyme catalyzes de novo strand initiation with GTP opposite a short cluster of C residues in the CCCA 3Ј termini that are a feature of both positive and negative strands of almost all amplifiable templates described in the literature (24,26,40). After fulllength transcription, termination is accompanied by the addition of a nontemplated A residue (6), thereby restoring the 3Ј A that was not copied into the complementary strand.…”

mentioning

confidence: 99%

“…Further, these elements are not universally present in the wide variety of short RNAs amplifiable by Q␤ replicase (40). Indeed, it was recognized several years ago (26) that the only feature common to replicatable RNAs appears to be the CCCA 3Ј terminus; this observation has held true with two exceptions, one being a variant Q␤ positive-sense RNA with a UCCA 3Ј end (28), the other being a 6S RNA amplified by Q␤ replicase with a GCCA terminus (33).…”

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confidence: 99%

Autonomous Role of 3′-Terminal CCCA in Directing Transcription of RNAs by Qβ Replicase

Tretheway¹,

Yoshinari²,

Dreher

2001

J Virol

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We have studied transcription in vitro by Q␤ replicase to deduce the minimal features needed for efficient end-to-end copying of an RNA template. Our studies have used templates ca. 30 nucleotides long that are expected to be free of secondary structure, permitting unambiguous analysis of the role of template sequence in directing transcription. A 3-terminal CCCA (3-CCCA) directs transcriptional initiation to opposite the underlined C; the amount of transcription is comparable between RNAs possessing upstream (CCA) n tracts, A-rich sequences, or a highly folded domain and is also comparable in single-round transcription assays to transcription of two amplifiable RNAs. Predominant initiation occurs within the 3-CCCA initiation box when a wide variety of sequences is present immediately upstream, but CCA or a closely similar sequence in that position results in significant internal initiation. Removal of the 3-A from the 3-CCCA results in 5-to 10-fold-lower transcription, emphasizing the importance of the nontemplated addition of 3-A by Q␤ replicase during termination. In considering whether 3-CCCA could provide sufficient specificity for viral transcription, and consequently amplification, in vivo, we note that tRNA His is the only stable Escherichia coli RNA with 3-CCCA. In vitro-generated transcripts corresponding to tRNA His served as poor templates for Q␤ replicase; this was shown to be due to the inaccessibility of the partially base-paired CCCA. These studies demonstrate that 3-CCCA plays a major role in the control of transcription by Q␤ replicase and that the abundant RNAs present in the host cell should not be efficient templates.Genome replication among the positive-strand RNA viruses is accomplished by sequential end-to-end transcriptions, first of the encapsidated positive sense RNA and subsequently of the newly synthesized negative-sense antigenome. Except for viruses whose genomes are covalently linked at the 5Ј end to a specialized protein, these transcriptions occur by de novo initiation (9). For successful replication by this pathway, transcriptional initiation must occur predominantly or exclusively at the 3Ј ends of genome and antigenome RNAs, with minimal initiation occurring internally or on other RNAs present within the cell. Q␤ replicase provides a convenient means to study the template properties underlying these required specificities for a representative positive-strand RNA virus.Q␤ replicase is the 4-subunit RNA-dependent RNA polymerase (RdRp) enzyme complex that amplifies the 4.2 kb positive-strand genome of bacteriophage Q␤, a phage infecting Escherichia coli (6,34). Unlike the RdRp of any eukaryotic positive-strand RNA virus, Q␤ replicase has been purified to homogeneity and shown to be capable of supporting the full viral genome amplification cycle in vitro (6). This enzyme catalyzes de novo strand initiation with GTP opposite a short cluster of C residues in the CCCA 3Ј termini that are a feature of both positive and negative strands of almost all amplifiable templates described in...

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Design of Artificial Short-Chained RNA Species That Are Replicated by Q.beta. Replicase

Cited by 39 publications

References 30 publications

Evolutionary reconstruction of a hairpin deleted from the genome of an RNA virus.

Evolutionary reconstruction of a hairpin deleted from the genome of an RNA virus.

Design of interacting multi-stable nucleic acids for molecular information processing

Autonomous Role of 3′-Terminal CCCA in Directing Transcription of RNAs by Qβ Replicase

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