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

The human RNA polymerase I structure reveals an HMG-like transcription factor docking domain specific to metazoans

2021 ◽  
Author(s):  
Julia L Daiß ◽  
Michael Pilsl ◽  
Kristina Straub ◽  
Andrea Bleckmann ◽  
Mona Höcherl ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Cellular Growth ◽  
Hmg Box ◽  
Transcription Bubble ◽  
Transcription System ◽  
Pol I ◽  
Additional Domain

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP-fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG-box incapable of DNA-binding which may serve as a downstream-transcription factor binding platform in metazoans. Biochemical analysis and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG-box domain containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

scholarly journals A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH and recombinant TBP, TFIIB, TFIIE and TFIIF

2001 ◽  
Vol 268 (16) ◽  
pp. 4527-4536 ◽  
Author(s):  
Irina Kotova ◽  
Anna Lena Chabes ◽  
Bo Segerman ◽  
Sara Flodell ◽  
Lars Thelander ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Transcription System

scholarly journals A majority ofRhodobacter sphaeroidespromoters lack a crucial RNA polymerase recognition feature, enabling coordinated transcription activation

2020 ◽  
Vol 117 (47) ◽  
pp. 29658-29668
Author(s):  
Kemardo K. Henry ◽  
Wilma Ross ◽  
Kevin S. Myers ◽  
Kimberly C. Lemmer ◽  
Jessica M. Vera ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Stationary Phase ◽  
Bacterial Species ◽  
Bioinformatic Analysis ◽  
In Vitro Transcription ◽  
Rrna Synthesis ◽  
Transcription System ◽  
Bacterial Ribosome ◽  
Genome Wide

Using an in vitro transcription system with purified RNA polymerase (RNAP) to investigate rRNA synthesis in the photoheterotrophic α-proteobacteriumRhodobacter sphaeroides, we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription fromR. sphaeroidesrRNA promoters was unexpectedly weak, correlating with absence of −7T, the very highly conserved thymine found at the last position in −10 elements of promoters in most bacterial species. Thymine substitutions for adenine at position −7 in the three rRNA promoters strongly increased intrinsic promoter activity, indicating thatR. sphaeroidesRNAP can utilize −7T when present. rRNA promoters were activated by purifiedR. sphaeroidesCarD, a transcription factor found in many bacterial species but not in β- and γ-proteobacteria. Overall, CarD increased the activity of 15 of 16 nativeR. sphaeroidespromoters tested in vitro that lacked −7T, whereas it had no effect on three of the four native promoters that contained −7T. Genome-wide bioinformatic analysis of promoters fromR. sphaeroidesand two other α-proteobacterial species indicated that 30 to 43% contained −7T, whereas 90 to 99% of promoters from non–α-proteobacteria contained −7T. Thus, promoters lacking −7T appear to be widespread in α-proteobacteria and may have evolved away from consensus to enable their coordinated regulation by transcription factors like CarD. We observed a strong reduction inR. sphaeroidesCarD levels when cells enter stationary phase, suggesting that reduced activation by CarD may contribute to inhibition of rRNA transcription when cells enter stationary phase, the stage of growth when bacterial ribosome synthesis declines.

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 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.

scholarly journals In Vitro Transcription of pe38/Polyhedrin Hybrid Promoters Reveals Sequences Essential for Recognition by the Baculovirus-Induced RNA Polymerase and for the Strength of Very Late Viral Promoters

1998 ◽  
Vol 72 (4) ◽  
pp. 2991-2998 ◽  
Author(s):  
Ruud M. W. Mans ◽  
Dagmar Knebel-Mörsdorf
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Sequence Motif ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Polyhedrin Promoter ◽  
Viral Promoters ◽  
Nuclear Polyhedrosis ◽  
Hybrid Promoters

ABSTRACT In vitro transcription was used to analyze the promoter specificity of the α-amanitin-resistant RNA polymerase that is induced late during infection of Autographa californica multicapsid nuclear polyhedrosis virus. By modifying the preparation of crude nuclear extracts, we have established an assay that permits differentiation between weak late and strong very late viral promoters. The virus-induced RNA polymerase initiates at a TAAG sequence motif in both late and very late promoters. Based on the sensitivity of our in vitro transcription system, we have investigated the sequences responsible for a functional TAAG motif and their putative role with respect to the strength of very late promoters. By constructing hybrid promoters between the early pe38 and the very late polyhedrin promoters, we demonstrated that the replacement of 7 nucleotides upstream of the nonfunctional TAAG sequences in the pe38 promoter with the corresponding sequences of the polyhedrin promoter was sufficient for recognition by the virus-induced RNA polymerase. The strength of the very late polyhedrin promoter was established after replacing the 5′ untranslated sequences of the pe38 promoter by those of the polyhedrin promoter in addition to the 7 nucleotides upstream of the TAAG motif.

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