In vitro transcription of viroid RNA into full-length copies by RNA-dependent RNA polymerase from healthy tomato leaf tissue

1982 ◽  
Vol 2 (3) ◽  
pp. 185-194 ◽  
Author(s):  
Frank Boege ◽  
Wolfgang Rohde ◽  
Heinz L. Sänger
Keyword(s):  
Rna Polymerase ◽  
Tomato Plant ◽  
Leaf Tissue ◽  
Potato Spindle Tuber Viroid ◽  
In Vitro Transcription ◽  
Tomato Leaf ◽  
Full Length ◽  
Rna Dependent Rna Polymerase ◽  
In Vitro Synthesis

RNA-dependent RNA polymerase from healthy tomato plant tissue accepts potato spindle tuber viroid (PSTV) RNA as a template for the in vitro synthesis of full-length RNA copies of the PSTV genome. Viroid transcription requires the presence of Mn2+ and/or Mg2+ ions and is not inhibited by concentrations of 10−5 M α-amanitin. This is the first report of a well-defined product synthesized in vitro by an RNA-dependent RNA polymerase from healthy plants.

scholarly journals Perturbation in the Conserved Methyltransferase-Polymerase Interface of Flavivirus NS5 Differentially Affects Polymerase Initiation and Elongation

2014 ◽  
Vol 89 (1) ◽  
pp. 249-261 ◽  
Author(s):  
Jiqin Wu ◽  
Guoliang Lu ◽  
Bo Zhang ◽  
Peng Gong
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Full Length ◽  
Intramolecular Interactions ◽  
Rna Dependent Rna Polymerase ◽  
Hydrophobic Residues ◽  
Functional Relevance

ABSTRACTThe flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through ade novomechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intramolecular interactions remain elusive. By solving the crystal structure of the Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal first 261 residues and other residues up to the first 303 residues deleted. Compared to the wild-type (WT) NS5, full-length NS5 variants exhibited consistent up- or downregulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay, from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266 to 275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276 to 303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface is relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis.IMPORTANCEThe flavivirus NS5 is very unique in having a methyltransferase (MTase) placed on the immediate N terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5, which revealed a conserved interface between MTase and RdRP. Building on this discovery, here we carried outin vitropolymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitions. By comparing the N-terminal-truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally unclarified previously. Taken together, we provide biochemical evidence and mechanistic insights for the cross talk between the two enzyme modules of flavivirus NS5.

scholarly journals Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA

1998 ◽  
Vol 72 (11) ◽  
pp. 8913-8920 ◽  
Author(s):  
Kun Yao ◽  
Vikram N. Vakharia
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
In Vitro Transcription ◽  
Infectious Pancreatic Necrosis Virus ◽  
Rna Dependent Rna Polymerase ◽  
Rna Transcripts ◽  
Recovered Virus ◽  
Cloned Cdna ◽  
Pancreatic Necrosis Virus

ABSTRACT We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of theBirnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3′-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3′ end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

Establishment of in vitro transcription system based on RNA-dependent RNA polymerase (RdRp) of Little cherry virus 1

2021 ◽  
Vol 65 (01) ◽  
pp. 101-103
Author(s):  
Xinran Cao ◽  
Yalan Wang ◽  
Guowei Geng ◽  
Yifan Sun ◽  
Chenmeng Gao ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Rna Dependent Rna Polymerase ◽  
Transcription System

Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I

1985 ◽  
Vol 5 (6) ◽  
pp. 1358-1369
Author(s):  
R M Learned ◽  
S Cordes ◽  
R Tjian
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Rna Polymerase I ◽  
Control Element ◽  
In Vitro Synthesis ◽  
Transcription System ◽  
Activation Of Transcription ◽  
Promoter Recognition ◽  
Polymerase I

A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have shown that the addition of SL1 can reprogram the otherwise nonpermissive mouse transcription system to recognize and initiate accurate RNA synthesis from human rDNA. Antibodies raised against SL1 bind preferentially to a protein localized in the nucleolus of primate cells and specifically inhibit in vitro transcription initiating from the human rRNA promoter. By contrast, anti-SL1 does not react with the nucleolus of rodent cells and has no effect on the in vitro synthesis of mouse rRNA by a transcription system derived from mouse cells. These findings suggest that SL1 is a selectivity factor present in the nucleolus that imparts promoter recognition to RNA polymerase I and that can discriminate between rRNA promoters from different species.

scholarly journals Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I.

1985 ◽  
Vol 5 (6) ◽  
pp. 1358-1369 ◽  
Author(s):  
R M Learned ◽  
S Cordes ◽  
R Tjian
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Rna Polymerase I ◽  
Control Element ◽  
In Vitro Synthesis ◽  
Transcription System ◽  
Activation Of Transcription ◽  
Promoter Recognition ◽  
Polymerase I

A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have shown that the addition of SL1 can reprogram the otherwise nonpermissive mouse transcription system to recognize and initiate accurate RNA synthesis from human rDNA. Antibodies raised against SL1 bind preferentially to a protein localized in the nucleolus of primate cells and specifically inhibit in vitro transcription initiating from the human rRNA promoter. By contrast, anti-SL1 does not react with the nucleolus of rodent cells and has no effect on the in vitro synthesis of mouse rRNA by a transcription system derived from mouse cells. These findings suggest that SL1 is a selectivity factor present in the nucleolus that imparts promoter recognition to RNA polymerase I and that can discriminate between rRNA promoters from different species.

scholarly journals RNA-dependent RNA polymerase from healthy tomato leaf tissue

FEBS Letters ◽  
1980 ◽  
Vol 121 (1) ◽  
pp. 91-96 ◽  
Author(s):  
Frank Boege ◽  
L.Sänger Heinz
Keyword(s):  
Rna Polymerase ◽  
Leaf Tissue ◽  
Tomato Leaf ◽  
Rna Dependent Rna Polymerase
Sign in / Sign up

Export Citation Format

Share Document