DNA-dependent RNA polymerase from bacteriophage T3 transcribes and amplifies an RNA template in vitro

Leary, Susan L.; Baum, Howard; Loewy, Zvi G.

doi:10.1016/0378-1119(91)90570-2

Cited by 9 publications

(5 citation statements)

References 6 publications

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“…Other functions that are unique to the particular type of poiymerase (e.g., promoter recognition and binding for the RNAPs, proofreading and exonuclease functions for the DNAPs) are iikeiy to be located elsewhere in the polymerase, possibly in auxiliary domains. The observations that T3 RNAP can utilize RNA as a tempiate (Leary etal,, 1991) and that T7 RNAP can replicate small RNA molecules (Konarska and Sharp, 1989) and incorporate deoxyribonucleotides (Wyatt and Walker, 1989) further obscure the distinctions among these nucleotide polymerases.…”

Section: The Simpie Nucieotide Polymerases Are Likely To Have a Modulmentioning

confidence: 99%

The phage RNA polymerases are related to DNA polymerases and reverse transcriptases

McAllister

Raskin

1993

Molecular Microbiology

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The single subunit DNA-dependent RNA polymerase (RNAP) that is encoded by bacteriophage T7 is the prototype of a class of relatively simple RNAPs that includes the RNAPs of the related phages T3 and SP6, as well as the mitochondrial RNAPs. The T7 enzyme has been crystallized, and recent genetic and biochemical analyses have facilitated an interpretation of this structure. A growing body of evidence suggests that the phage-like RNAPs are related to other nucleotide polymerases such as DNA polymerases, RNA-dependent RNA polymerases, and reverse transcriptases. In this work, we review information concerning the structure and function of T7 RNAP, and evidence in support of its assignment to a broader class of nucleotide polymerases.

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Section: The Simpie Nucieotide Polymerases Are Likely To Have a Modulmentioning

confidence: 99%

The phage RNA polymerases are related to DNA polymerases and reverse transcriptases

McAllister

Raskin

1993

Molecular Microbiology

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“…Many of these previous studies examined the phage-encoded SP6 RNA polymerase (SP6RP) (7,9,26,27,30,41). SP6RP has been used to produce synthetic RNA for a wide variety of applications (29,30,33,37,43).…”

mentioning

confidence: 99%

Complete Genomic Sequence of the Virulent Salmonella Bacteriophage SP6

et al. 2004

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We report the complete genome sequence of enterobacteriophage SP6, which infects Salmonella enterica serovar Typhimurium. The genome contains 43,769 bp, including a 174-bp direct terminal repeat. The gene content and organization clearly place SP6 in the coliphage T7 group of phages, but there is ϳ5 kb at the right end of the genome that is not present in other members of the group, and the homologues of T7 genes 1.3 through 3 appear to have undergone an unusual reorganization. Sequence analysis identified 10 putative promoters for the SP6-encoded RNA polymerase and seven putative rho-independent terminators. The terminator following the gene encoding the major capsid subunit has a termination efficiency of about 50% with the SP6-encoded RNA polymerase. Phylogenetic analysis of phages related to SP6 provided clear evidence for horizontal exchange of sequences in the ancestry of these phages and clearly demarcated exchange boundaries; one of the recombination joints lies within the coding region for a phage exonuclease. Bioinformatic analysis of the SP6 sequence strongly suggested that DNA replication occurs in large part through a bidirectional mechanism, possibly with circular intermediates.Bacteriophage SP6 is a small double-stranded DNA tailed phage that infects Salmonella enterica serovar Typhimurium LT2. It shares many features with coliphage T7; however, no significant DNA sequence homology was detected between these phages by DNA hybridization (26). Previous studies of SP6 have elucidated its virion structure and the function and regulation of its early genes. Many of these previous studies examined the phage-encoded SP6 RNA polymerase (SP6RP) (7,9,26,27,30,41). SP6RP has been used to produce synthetic RNA for a wide variety of applications (29,30,33,37,43).As with phage T7, the SP6 genome is transcribed in a temporally ordered manner (26); transcription by SP6RP gives rise to 10 discrete RNA species (26). This finding suggests that discrete sites present in the SP6 genome serve as specific initiation and termination signals. One terminator sequence for the SP6 species IX RNA transcript has been identified, cloned, and sequenced (5). This sequence appears to be analogous to the stem-loop structure found at a comparable position in the T7 genome and to other rho-independent terminators (14). Preliminary studies have provided evidence for the presence of this termination sequence in the SP6 genome (5), but the termination efficiency has not been fully characterized.Bacteriophage SP6 has been reported to be closely related to phages K1-5, K5, and K1E (51). In addition, Scholl et al. reported that SP6 encodes a tail protein that is in the same family as the tail spike protein of the otherwise apparently unrelated phage P22 (51). However, information about SP6 phage genetics, SP6 molecular biology, and the relationship of the sequence of this phage to other phage and prophage sequences is still limited. We completed the 43,769-bp sequence of the phage SP6 genome, identified the positions of individual genes an...

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“…It was of interest to compare the T7 RNAP protein with the two other RNAP proteins commonly used for in vitro transcription purposes, the SP6 and T3 RNAPs (12)(13)(14). To this end, the T7 RNAP gene in R011 was replaced with either the SP6 or the T3 RNAP gene.…”

Section: Autogene Expression Is Not Sensitive To the Type Of Phage Rnmentioning

confidence: 99%

Factors limiting autogene‐based cytoplasmic expression systems

Finn

MacLachlan²,

Cullis

2005

FASEB j.

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The relatively low levels of transfection that can be achieved by current gene delivery systems have limited the therapeutic utility of gene transfer. This is especially true for non-viral gene delivery systems, where the levels of gene expression achieved are usually well below the levels achieved by viral gene transfer systems. Previous work from our laboratory describes an enhanced dual promoter autogene-based cytoplasmic expression system that gives rise to levels of gene expression 20-fold higher than that of a CMV nuclear expression plasmid control. Here various strategies are described to increase the levels of autogene-based gene expression by changing variables such as the type of nuclear promoter, phage RNAP gene, and IRES element. Although insights into the function of various IRES elements were gained, none of these changes demonstrated a significant increase in gene expression. However, determination of the mRNA levels achieved using quantitative RNase protection assays and immunofluorescence experiments revealed that transgene mRNA levels were saturated at up to 10 times higher than all other mRNA in the transfected cell combined. It follows that mRNA production, as well as translation, are important factors limiting autogene-based cytoplasmic expression.

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DNA-dependent RNA polymerase from bacteriophage T3 transcribes and amplifies an RNA template in vitro

Cited by 9 publications

References 6 publications

The phage RNA polymerases are related to DNA polymerases and reverse transcriptases

The phage RNA polymerases are related to DNA polymerases and reverse transcriptases

Complete Genomic Sequence of the Virulent Salmonella Bacteriophage SP6

Factors limiting autogene‐based cytoplasmic expression systems

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