Abortive initiation by bacteriophage T3 and T7 RNA polymerases under conditions of limiting substrate

Ling, Mei-Ling; Risman, Stevens S.; Klement, John F.; McGraw, Nancy; McAllister, William T.

doi:10.1093/nar/17.4.1605

Cited by 61 publications

(33 citation statements)

References 20 publications

(19 reference statements)

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“…To overcome this problem, we inserted either one or two additional G residues at the transcription initiation site in the PaV1 plasmid to improve the sequence context of the T7 promoter and thereby enhance the level of DNA-templated primary transcription (33). An additional G residue at the initiation site for T7 transcription was also found necessary for replication initiated by plasmids containing FHV1 cDNA (3).…”

Section: Resultsmentioning

confidence: 99%

Recovery of Infectious Pariacoto Virus from cDNA Clones and Identification of Susceptible Cell Lines

Johnson

Ball

2001

J Virol

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Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T‫3؍‬ icosahedral symmetry. The 3.0-Å crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor ␣. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from Spodoptera exigua, Helicoverpa zea and Aedes albopictus, whereas cells from Drosophila melanogaster and Spodoptera frugiperda were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from Helicoverpa zea (corn earworm) and for larvae of Galleria mellonella (greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly.Members of the Nodaviridae family are small positive-sense RNA viruses with Tϭ3 icosahedral symmetry (for a review, see references 4, 5, and 37). Each 30-nm particle is assembled from 180 copies of the capsid protein precursor ␣ and one copy of each of the two unique segments of the viral RNA genome (24,30,41). Viruses from the alphanodavirus genus infect primarily insects. In alphanodaviruses, the 44-kDa capsid protein precursor ␣ is autocatalytically cleaved following virion assembly to yield the two mature capsid proteins ␤ (40 kDa) and ␥ (4 kDa) (20). The maturation cleavage is required for infectivity (38). The larger genome segment (RNA1) contains 3.0 to 3.2 kb and encodes protein A, the catalytic subunit of the RNAdependent RNA polymerase (26). The smaller genome segment (RNA2) contains 1.3 to 1.4 kb and encodes the capsid protein precursor, ␣. Both genomic RNAs are capped but not polyadenylated (9,10,22,28). During replication, a subgenomic RNA (RNA3) which is 3Ј coterminal with RNA1 is transcribed. RNA3 contains about 400 nucleotides (nt) and encodes one or two small proteins (B1 and B2) of unknown function (5,16,22).The best studied alphanodavirus is Flock House virus (FHV), and reverse genetic systems that allow the infectious cycle of this virus to be reconstructed from cDNA clones have been developed. FHV replication can be initiated and infectious virus can be recovered from in vitro transcripts of RNA1 an...

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Section: Resultsmentioning

confidence: 99%

Recovery of Infectious Pariacoto Virus from cDNA Clones and Identification of Susceptible Cell Lines

Johnson

Ball

2001

J Virol

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“…During the early stages of transcription, T7 RNAP engages in multiple cycles of initiation in which short RNA products are synthesized and released without movement of the RNAP away from the promoter (5)(6)(7)(8)(9). Once the enzyme has initiated transcription and cleared the promoter, the ternary elongation complex is highly processive and is able to synthesize long RNA chains (Ͼ15,000 nt) until it encounters a termination signal or reaches the end of the template.…”

Section: T7 Rna Polymerase (Rnap)mentioning

confidence: 99%

“…ent with the remarkably low stability of rU:dA base pairs (27), the decreased yield of product is more difficult to explain. It has been observed that during the early stages of initiation T7 RNAP aborts transcription more frequently before or after incorporating a UMP residue, and it has been suggested that the weak interactions of the rU:dA base pair may hinder the correct orientation of the base in the active site, resulting in slower catalysis (5,8). Thus, even though dissociation of the transcription complex would be enhanced when transcribing through a poly(dA) tract, the increased time required for the polymerase to reach the point of release would result in a slower turnover rate and hence a lower yield.…”

Section: Figmentioning

confidence: 99%

Template Strand Switching by T7 RNA Polymerase

Rong¹,

Durbin²,

McAllister

1998

Journal of Biological Chemistry

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T7 RNA polymerase (RNAP) is able to traverse a variety of discontinuities in the template (T) strand of duplex DNA, including nicks, gaps, and branched junctions in which the 3 end of the T strand is not complementary to the non-template (NT) strand. The products represent a faithful copy of the T strand, with no insertions or deletions. On double-stranded templates having protruding 3 ends the polymerase is able to insert the free 3 end of the NT strand and to utilize this as a new T strand ("turn around transcription"), resulting in the anomalous production of high molecular weight transcripts.The capacity of T7 RNAP to bypass interruptions in the T strand depends upon the stability of the elongation complex. Sequences that are expected to stabilize a local RNA:DNA hybrid (such as the presence of a C 6 tract in the T strand) dramatically reduce dissociation of the RNAP while still allowing the enzyme to insert a new 3 end. Similar effects on RNAP release are observed when the enzyme reaches the end of a template (i.e. when synthesizing runoff products), resulting in markedly different yields of RNA product during multiple rounds of transcription. T7 RNA polymerase (RNAP)1 is a single subunit enzyme that is able to carry out all of the steps in the transcription process without the need for auxiliary factors (for review see Ref. 1). The crystal structure of the enzyme has been solved to a resolution of 3.3 Å (2), and it is therefore highly attractive as a model for studies of the mechanism of RNA synthesis (for review see Refs. 3 and 4).During the early stages of transcription, T7 RNAP engages in multiple cycles of initiation in which short RNA products are synthesized and released without movement of the RNAP away from the promoter (5-9). Once the enzyme has initiated transcription and cleared the promoter, the ternary elongation complex is highly processive and is able to synthesize long RNA chains (Ͼ15,000 nt) until it encounters a termination signal or reaches the end of the template. The transition from an unstable initiation complex to a stable elongation complex (EC) is accompanied by isomerization of the complex (6, 10, 11) and may involve wrapping of a flexible "thumb" domain (12, 13) and possibly a "specificity loop" (1, 14) around one or both strands of the template, leading to partial closure of the DNA binding cleft.The non-template (NT) strand of the DNA does not appear to be required for initiation or for the early stages of transcription, as Maslak and Martin (15) have shown that T7 RNAP can initiate transcription at a promoter that is single-stranded in the initiation region (from Ϫ6 and downstream), and Milligan et al. (7) have shown that the enzyme can transcribe a singlestranded DNA template once it has initiated transcription from a double-stranded promoter. However, Zhou et al. (16,17) have suggested that the stability of the EC may require the presence of duplex DNA at the leading edge of the elongation complex, as an elongation complex that is halted just upstream from a region in which ...

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“…A variety of experimental results indicate that the binding region is recognized as a double strand duplex upstream from Ϫ6 and that the initiation region is melted open very rapidly upon (or simultaneously with) polymerase binding (7)(8)(9)(10)(11). During the early stages of transcription, T7 RNAP engages in repeated cycles of abortive initiation in which short RNA products are synthesized and released before the polymerase clears the promoter and forms a stable elongation complex (12)(13)(14). Footprinting studies with methidiumpropyl EDTA-Fe(II) have shown that the polymerase protects the promoter as far upstream as Ϫ21 during this process and that these contacts are maintained until the polymerase isomerizes into a processive elongation complex (15).…”

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confidence: 99%

Promoter specificity determinants of T7 RNA polymerase

Rong

He²,

McAllister³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742-773) that projects into the DNA binding cleft (1). Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions ؊10 and ؊11 (2). A comparison of the sequences of other phage RNAPs and their promoters suggested additional contacts that might be important in promoter recognition. We have found that changing the amino acid residue at position 758 in T7 RNAP results in an enzyme with altered specificity for the bp at position ؊8. The identification of two amino acid:base pair contacts (i.e., N748 with the bp at ؊10 and ؊11, and Q758 with the bp at ؊8) provides information concerning the disposition of the specificity loop relative to the upstream region of the promoter. The results suggest that substantial rearrangements of the loop (and͞or the DNA) are likely to be required to allow these amino acids to interact with their cognate base pairs during promoter recognition.The single subunit DNA-dependent RNA polymerases (RNAPs) that are encoded by bacteriophage T7 and its relatives (e.g., T3, SP6, K11) are highly specific for their individual promoter sequences (for review, see ref.3). Although each promoter consensus sequence is related to a common sequence that extends from Ϫ17 to ϩ6, significant differences in the interval from Ϫ8 to Ϫ11 suggest that this region may be critical to the discrimination of the promoter by its respective RNAP (Fig. 1). Indeed, in earlier work the bp at Ϫ10 and Ϫ11 were found to be the primary determinants of specificity for T7 versus T3 promoters, and the bp at Ϫ8 and Ϫ9 were found to be the primary determinants of specificity for SP6 versus T7 promoters (4, 5).Promoters for the phage RNAP seem to consist of two functional domains: a binding domain that extends from Ϫ17 to Ϫ6 and an initiation domain that extends from Ϫ5 to ϩ6 (6, 7). In general, variations in the binding domain affect the affinity of the RNAP for the promoter but have little effect on initiation (k cat ), whereas variations in the initiation region affect k cat but have little effect on binding (6, 7). A variety of experimental results indicate that the binding region is recognized as a double strand duplex upstream from Ϫ6 and that the initiation region is melted open very rapidly upon (or simultaneously with) polymerase binding (7-11). During the early stages of transcription, T7 RNAP engages in repeated cycles of abortive initiation in which short RNA products are synthesized and released before the polymerase clears the promoter and forms a stable elongation complex (12)(13)(14). Footprinting studies with methidiumpropyl EDTA-Fe(II) have shown that the polymerase protects the promoter as far upstream as Ϫ21 during this process and that these contacts are maintained until the polymerase isomerizes into a processive elongation complex (15).The topology of T7 RNAP:promoter contacts in the bi...

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Abortive initiation by bacteriophage T3 and T7 RNA polymerases under conditions of limiting substrate

Cited by 61 publications

References 20 publications

Recovery of Infectious Pariacoto Virus from cDNA Clones and Identification of Susceptible Cell Lines

Recovery of Infectious Pariacoto Virus from cDNA Clones and Identification of Susceptible Cell Lines

Template Strand Switching by T7 RNA Polymerase

Promoter specificity determinants of T7 RNA polymerase

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