scholarly journals Ribozyme-catalysed RNA synthesis using triplet building blocks

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
James Attwater ◽  
Aditya Raguram ◽  
Alexey S Morgunov ◽  
Edoardo Gianni ◽  
Philipp Holliger
Keyword(s):  
Rna Synthesis ◽  
Rna Folding ◽  
Rna Replication ◽  
Building Blocks ◽  
Secondary Structures ◽  
Catalytic Subunit ◽  
Rna Catalysis ◽  
Apparent Paradox ◽  
In Vitro Evolution

RNA-catalyzed RNA replication is widely believed to have supported a primordial biology. However, RNA catalysis is dependent upon RNA folding, and this yields structures that can block replication of such RNAs. To address this apparent paradox, we have re-examined the building blocks used for RNA replication. We report RNA-catalysed RNA synthesis on structured templates when using trinucleotide triphosphates (triplets) as substrates, catalysed by a general and accurate triplet polymerase ribozyme that emerged from in vitro evolution as a mutualistic RNA heterodimer. The triplets cooperatively invaded and unraveled even highly stable RNA secondary structures, and support non-canonical primer-free and bidirectional modes of RNA synthesis and replication. Triplet substrates thus resolve a central incongruity of RNA replication, and here allow the ribozyme to synthesise its own catalytic subunit ‘+’ and ‘–’ strands in segments and assemble them into a new active ribozyme.

scholarly journals Relaxed Substrate Specificity in Qβ Replicase through Long-Term In Vitro Evolution

Life ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 32
Author(s):  
Kohtoh Yukawa ◽  
Ryo Mizuuchi ◽  
Norikazu Ichihashi
Keyword(s):  
Substrate Specificity ◽  
Early Life ◽  
Evolutionary History ◽  
Rna Replication ◽  
Life Forms ◽  
In Vitro Evolution ◽  
Present Evidence ◽  
Major Transition

A change from RNA- to DNA-based genetic systems is hypothesized as a major transition in the evolution of early life forms. One of the possible requirements for this transition is a change in the substrate specificity of the replication enzyme. It is largely unknown how such changes would have occurred during early evolutionary history. In this study, we present evidence that an RNA replication enzyme that has evolved in the absence of deoxyribonucleotide triphosphates (dNTPs) relaxes its substrate specificity and incorporates labeled dNTPs. This result implies that ancient replication enzymes, which probably evolved in the absence of dNTPs, could have incorporated dNTPs to synthesize DNA soon after dNTPs became available. The transition from RNA to DNA, therefore, might have been easier than previously thought.

scholarly journals Differential Rescue of Poliovirus RNA Replication Functions by Genetically Modified RNA Polymerase Precursors

2004 ◽  
Vol 78 (23) ◽  
pp. 13007-13018 ◽  
Author(s):  
Christopher T. Cornell ◽  
Jo Ellen Brunner ◽  
Bert L. Semler
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Genetically Modified ◽  
Rna Replication ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Wild Type ◽  
Positive Strand ◽  
Positive Strand Rna

ABSTRACT We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

scholarly journals Mechanism of Stimulation of Plus-Strand Synthesis by an RNA Replication Enhancer in a Tombusvirus

2005 ◽  
Vol 79 (15) ◽  
pp. 9777-9785 ◽  
Author(s):  
Tadas Panavas ◽  
Peter D. Nagy
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Replication ◽  
Dual Function ◽  
Long Distance ◽  
Replication Assay ◽  
Rna Interaction ◽  
Stimulation Of

ABSTRACT Replication of RNA viruses is regulated by cis-acting RNA elements, including promoters, replication silencers, and replication enhancers (REN). To dissect the function of an REN element involved in plus-strand RNA synthesis, we developed an in vitro trans-replication assay for tombusviruses, which are small plus-strand RNA viruses. In this assay, two RNA strands were tethered together via short complementary regions with the REN present in the nontemplate RNA, whereas the promoter was located in the template RNA. We found that the template activity of the tombusvirus replicase preparation was stimulated in trans by the REN, suggesting that the REN is a functional enhancer when located in the vicinity of the promoter. In addition, this study revealed that the REN has dual function during RNA synthesis. (i) It binds to the viral replicase. (ii) It interacts with the core plus-strand initiation promoter via a long-distance RNA-RNA interaction, which leads to stimulation of initiation of plus-strand RNA synthesis by the replicase in vitro. We also observed that this RNA-RNA interaction increased the in vivo accumulation and competitiveness of defective interfering RNA, a model template. We propose that REN is important for asymmetrical viral RNA replication that leads to more abundant plus-strand RNA progeny than the minus-strand intermediate, a hallmark of replication of plus-strand RNA viruses.

scholarly journals Identification of an RNA Hairpin in Poliovirus RNA That Serves as the Primary Template in the In Vitro Uridylylation of VPg

2000 ◽  
Vol 74 (22) ◽  
pp. 10359-10370 ◽  
Author(s):  
Aniko V. Paul ◽  
Elizabeth Rieder ◽  
Dong Wook Kim ◽  
Jacques H. van Boom ◽  
Eckard Wimmer
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Covalent Attachment ◽  
Minus Strand ◽  
Coding Region ◽  
Rna Sequences ◽  
Cellular Factors ◽  
Rna Hairpin ◽  
Made In

ABSTRACT The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3Dpol, and Mg2+ (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280–284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590–4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CDpro. Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.

scholarly journals Structural and Functional Studies of the Promoter Element for Dengue Virus RNA Replication

2008 ◽  
Vol 83 (2) ◽  
pp. 993-1008 ◽  
Author(s):  
María F. Lodeiro ◽  
Claudia V. Filomatori ◽  
Andrea V. Gamarnik
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Polymerase Activity ◽  
Stem Loop ◽  
Denv Serotypes ◽  
Functional Studies ◽  
Promoter Recognition

ABSTRACT The 5′ untranslated region (5′UTR) of the dengue virus (DENV) genome contains two defined elements essential for viral replication. At the 5′ end, a large stem-loop (SLA) structure functions as the promoter for viral polymerase activity. Next to the SLA, there is a short stem-loop that contains a cyclization sequence known as the 5′ upstream AUG region (5′UAR). Here, we analyzed the secondary structure of the SLA in solution and the structural requirements of this element for viral replication. Using infectious DENV clones, viral replicons, and in vitro polymerase assays, we defined two helical regions, a side stem-loop, a top loop, and a U bulge within SLA as crucial elements for viral replication. The determinants for SLA-polymerase recognition were found to be common in different DENV serotypes. In addition, structural elements within the SLA required for DENV RNA replication were also conserved among different mosquito- and tick-borne flavivirus genomes, suggesting possible common strategies for polymerase-promoter recognition in flaviviruses. Furthermore, a conserved oligo(U) track present downstream of the SLA was found to modulate RNA synthesis in transfected cells. In vitro polymerase assays indicated that a sequence of at least 10 residues following the SLA, upstream of the 5′UAR, was necessary for efficient RNA synthesis using the viral 3′UTR as template.

scholarly journals Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

1998 ◽  
Vol 72 (8) ◽  
pp. 6546-6553 ◽  
Author(s):  
Julie A. Lemm ◽  
Anders Bergqvist ◽  
Carol M. Read ◽  
Charles M. Rice
Keyword(s):  
Rna Polymerase ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Functional Assay ◽  
Minus Strand ◽  
Virus Rna ◽  
A Genome ◽  
Strand Initiation

ABSTRACT Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

scholarly journals Structural basis for the function of SuhB as a transcription factor in ribosomal RNA synthesis

2019 ◽  
Vol 47 (12) ◽  
pp. 6488-6503 ◽  
Author(s):  
Yong-Heng Huang ◽  
Nelly Said ◽  
Bernhard Loll ◽  
Markus C Wahl
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Rna Folding ◽  
In Vitro Transcription ◽  
Structural Basis ◽  
Macromolecular Crystallography ◽  
Rna Transcription ◽  
Signal Element

AbstractRibosomal RNA synthesis in Escherichia coli involves a transcription complex, in which RNA polymerase is modified by a signal element on the transcript, Nus factors A, B, E and G, ribosomal protein S4 and inositol mono-phosphatase SuhB. This complex is resistant to ρ-dependent termination and facilitates ribosomal RNA folding, maturation and subunit assembly. The functional contributions of SuhB and their structural bases are presently unclear. We show that SuhB directly binds the RNA signal element and the C-terminal AR2 domain of NusA, and we delineate the atomic basis of the latter interaction by macromolecular crystallography. SuhB recruitment to a ribosomal RNA transcription complex depends on the RNA signal element but not on the NusA AR2 domain. SuhB in turn is required for stable integration of the NusB/E dimer into the complex. In vitro transcription assays revealed that SuhB is crucial for delaying or suppressing ρ-dependent termination, that SuhB also can reduce intrinsic termination, and that SuhB-AR2 contacts contribute to these effects. Together, our results reveal functions of SuhB during ribosomal RNA synthesis and delineate some of the underlying molecular interactions.

scholarly journals Membrane Requirements for Uridylylation of the Poliovirus VPg Protein and Viral RNA Synthesis In Vitro

2003 ◽  
Vol 77 (21) ◽  
pp. 11408-11416 ◽  
Author(s):  
Mark H. Fogg ◽  
Natalya L. Teterina ◽  
Ellie Ehrenfeld
Keyword(s):  
Membrane Trafficking ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Rna ◽  
Chain Elongation ◽  
Electron Microscopic ◽  
Ribonucleoprotein Complexes ◽  
Cell Extracts ◽  
Infected Cells

ABSTRACT Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.

scholarly journals Requirements for Assembly of Poliovirus Replication Complexes and Negative-Strand RNA Synthesis

2001 ◽  
Vol 75 (8) ◽  
pp. 3841-3850 ◽  
Author(s):  
Natalya L. Teterina ◽  
Denise Egger ◽  
Kurt Bienz ◽  
David M. Brown ◽  
Bert L. Semler ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Internal Ribosomal Entry Site ◽  
Rna Replication ◽  
Viral Proteins ◽  
Encephalomyocarditis Virus ◽  
Minus Strand ◽  
Entry Site ◽  
Sequence Production ◽  
Positive Strand Rna

ABSTRACT HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5′ noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5′-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5′-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3′ end.

scholarly journals Restriction of poliovirus RNA replication in persistently infected nerve cells

2002 ◽  
Vol 83 (5) ◽  
pp. 1087-1093 ◽  
Author(s):  
Sophie Girard ◽  
Anne-Sophie Gosselin ◽  
Isabelle Pelletier ◽  
Florence Colbère-Garapin ◽  
Thérèse Couderc ◽  
...  
Keyword(s):  
Acute Phase ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Rna ◽  
Paralytic Poliomyelitis ◽  
Minus Strand ◽  
Relative Level ◽  
Persistently Infected ◽  
Cellular Factors

The aetiology of post-polio syndrome may involve persistence of poliovirus (PV) in the CNS. PV persists in the CNS of infected paralysed mice for over a year after the acute phase of paralytic poliomyelitis. However, infectious PV particles cannot be recovered from homogenates of CNS from paralysed mice after the acute phase of disease, indicating that PV replication is restricted. To identify the molecular mechanism by which PV replication is limited, PV RNA synthesis was analysed by estimating the relative level of genomic (plus-strand) and complementary (minus-strand) PV RNA in the CNS of persistently infected mice. PV RNA replication decreased during the 6 months following onset of paralysis, due mainly to inhibition of plus-strand RNA synthesis. Thus, restriction of PV RNA synthesis may contribute to persistence by limiting virus replication in the mouse CNS. Interestingly, viral RNA replication was similarly inhibited in neuroblastoma IMR-32 cell cultures persistently infected with PV. This in vitro model thus shows that cellular factors play a role in the inhibition of viral RNA synthesis.

Sign in / Sign up

Export Citation Format

Share Document