Asymmetric RNA synthesis in vitro: heterologous DNA-enzyme systems; E. coli RNA polymerase.

1. RNA was synthesized in vitro from a template of bacteriophage T4 DNA, in the presence of Mn2+. A comparison was made of the RNA synthesized by purified RNA polymerase from two sources, Micrococcus lysodeikticus and Escherichia coli; these are referred to as Micrococcus cRNA and E. coli cRNA respectively (where cRNA indicates RNA synthesized in vitro by using purified RNA polymerase and a DNA primer). 2. Both types of RNA were self-complementary as judged by resistance to digestion with ribonuclease after self-annealing, Micrococcus cRNA being more self-complementary (40%) than was E. coli cRNA (30%). The cRNA was found to be much less self-complementary if Mg2+ was present during RNA synthesis instead of Mn2+. 3. Micrococcus cRNA hybridized with a larger part of bacteriophage T4 DNA than did E. coli cRNA. The E. coli cRNA competed with only part (70%) of the Micrococcus cRNA in hybridization-competition experiments. It is concluded that more sequences of bacteriophage T4 DNA are transcribed by Micrococcus polymerase than by E. coli polymerase. 4. The RNA sequences synthesized by Micrococcus RNA polymerase but not by E. coli RNA polymerase are shown by hybridization competition to compete with specifically late bacteriophage T4 messenger RNA sequences. The relevance of this finding to the control of transcription is discussed. 5. In an Appendix, new methods are described for the analysis of hybridization-saturation and -competition experiments. Particular attention is paid to the effects produced if different RNA sequences are present at different relative concentrations. 6. By using cRNA isolated from an enzymically synthesized DNA–RNA hybrid, it is estimated that, of the DNA that is complementary to cRNA, only about half can become hybridized with cRNA under the experimental conditions used.

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NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble

eLife ◽

10.7554/elife.18096 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 36

Author(s):

Matti Turtola ◽

Georgiy A Belogurov

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Transcription Elongation ◽

Single Nucleotide ◽

E Coli ◽

Transcription Bubble ◽

Nucleotide Addition ◽

Nascent Rna ◽

Comprehensive Mapping

Universally conserved factors from NusG family bind at the upstream fork junction of transcription elongation complexes and modulate RNA synthesis in response to translation, processing, and folding of the nascent RNA. Escherichia coli NusG enhances transcription elongation in vitro by a poorly understood mechanism. Here we report that E. coli NusG slows Gre factor-stimulated cleavage of the nascent RNA, but does not measurably change the rates of single nucleotide addition and translocation by a non-paused RNA polymerase. We demonstrate that NusG slows RNA cleavage by inhibiting backtracking. This activity is abolished by mismatches in the upstream DNA and is independent of the gate and rudder loops, but is partially dependent on the lid loop. Our comprehensive mapping of the upstream fork junction by base analogue fluorescence and nucleic acids crosslinking suggests that NusG inhibits backtracking by stabilizing the minimal transcription bubble.

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Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

Journal of Virology ◽

10.1128/jvi.74.24.11671-11680.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11671-11680 ◽

Cited By ~ 49

Author(s):

T. A. M. Osman ◽

C. L. Hemenway ◽

K. W. Buck

Keyword(s):

Rna Polymerase ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Untranslated Region ◽

Rna Synthesis ◽

Central Core ◽

Minus Strand ◽

Stem Loop ◽

Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

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Primed abortive initiation of RNA synthesis by E. coli RNA polymerase on T7 DNA. Steady state kinetic studies

Nucleic Acids Research ◽

10.1093/nar/5.6.1919 ◽

1978 ◽

Vol 5 (6) ◽

pp. 1919-1932 ◽

Cited By ~ 22

Author(s):

W.J. Smagowicz ◽

K.H. Scheit

Keyword(s):

Steady State ◽

Rna Polymerase ◽

Rna Synthesis ◽

Kinetic Studies ◽

E Coli ◽

Steady State Kinetic ◽

State Kinetic ◽

Abortive Initiation

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Factors Required for the Uridylylation of the Foot-and-Mouth Disease Virus 3B1, 3B2, and 3B3 Peptides by the RNA-Dependent RNA Polymerase (3Dpol) In Vitro

Journal of Virology ◽

10.1128/jvi.79.12.7698-7706.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7698-7706 ◽

Cited By ~ 57

Author(s):

Arabinda Nayak ◽

Ian G. Goodfellow ◽

Graham J. Belsham

Keyword(s):

Rna Polymerase ◽

Disease Virus ◽

Rna Synthesis ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Coding Region ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Positive Strand Rna

ABSTRACT The 5′ terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3Dpol. To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3Dpol in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV cre has been identified previously to be within the 5′ untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV cre (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

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RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Viruses ◽

10.3390/v13091738 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1738

Author(s):

Alesia A. Levanova ◽

Eeva J. Vainio ◽

Jarkko Hantula ◽

Minna M. Poranen

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Rna Virus ◽

Polymerization Reaction ◽

Rna Dependent Rna Polymerase ◽

Independent Manner ◽

Nucleotidyl Transferase ◽

Rna Polymerization ◽

The Family

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

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Effect of DNA-interacting drugs on phage T7 RNA polymerase.

Acta Biochimica Polonica ◽

10.18388/abp.1998_4295 ◽

1998 ◽

Vol 45 (1) ◽

pp. 127-132 ◽

Cited By ~ 3

Author(s):

M Piestrzeniewicz ◽

K Studzian ◽

D Wilmańska ◽

G Płucienniczak ◽

M Gniazdowski

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Actinomycin D ◽

T7 Rna Polymerase ◽

Distamycin A ◽

E Coli ◽

Phage T7 ◽

Drug Dependent ◽

Dna Complex ◽

Kinetics Of

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

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