Positioning of the start site in the initiation of transcription by bacteriophage T7 RNA polymerase 1 1Edited by R. Ebright

Weston, Benjamin F.; Kuzmine, I; Martin, Craig T.

doi:10.1006/jmbi.1997.1199

Cited by 25 publications

(36 citation statements)

References 31 publications

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“…5). This situation is similar to the results obtained with the well-characterized T7 RNA polymerase, where the ϩ1C is not required for the proper positioning of the initiation site in the catalytic pocket of the T7 RNA polymerase (59,67). Weston et al (67) demonstrated that the distance between the core promoter elements and the initiation cytidylate could be increased through the addition of flexible linkers, although increased nucleotide misincorporation resulted.…”

Section: Discussionsupporting

confidence: 80%

Template Nucleotide Moieties Required for De Novo Initiation of RNA Synthesis by a Recombinant Viral RNA-Dependent RNA Polymerase

Kim

Zhong²,

Hong³

et al. 2000

J Virol

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Virology 253:1-7, 1999). Using RNAs containing nucleotide analogs, we found that the N3 and C4-amino group at the initiation cytidine were required for RNA synthesis. However, the ribose C2-hydroxyl of the initiating cytidylate can accept several modifications and retain the ability to direct synthesis. The only unacceptable modification is a protonated C2-amino group. Quite strikingly, the recognition of the functional groups for the initiation cytidylate and other template nucleotides are different. For example, a C5-methyl group in cytidine can direct RNA synthesis at all template positions except at the initiation cytidylate and C2-amino modifications are tolerated better after the ؉11 position. When a 4-thiouracil (4sU) base analog that allows only imperfect base pairing with the nascent RNA is placed at different positions in the template, the efficiency of synthesis is correlated with the calculated stability of the template-nascent RNA duplex adjacent to the position of the 4sU. These results define the requirements for the specific interactions required for the initiation of RNA synthesis and will be compared to the mechanisms of initiation by other RNA-dependent and DNA-dependent RNA polymerases.De novo (primer-independent) initiation is an important mechanism for viral RNA replication that requires the specific and appropriate interactions of at least four components: (i) an RNA template with a virus-specific initiation nucleotide at or near the 3Ј end; (ii) an initiation nucleoside triphosphate (NTP); (iii) a second NTP; and (iv) an RNA-dependent RNA polymerase (RdRp). Improper recognition may reduce or inhibit efficient RNA synthesis. Little is known about specific recognition between RdRp and RNA template moieties for de novo initiation of viral RNA synthesis. Furthermore, the interactions may change as the polymerase undergoes conformational changes during synthesis (11,32).Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus in the Flaviviridae family, which includes human and animal pathogens, such as Hepatitis C virus (HCV), Dengue virus, and Yellow fever virus (10,34,55). BVDV is an enveloped virus containing a single-stranded positive-sense RNA genome (55). In addition to being an important pathogen of livestock (51), BVDV is a model system for HCV infection and for building chimeric viruses with HCV (14,31,61,68).The BVDV RNA genome is translated as a polyprotein through a cap-independent mechanism after infection. The polyprotein is further processed into individual structural and nonstructural proteins by a combination of cellular and viral proteases (10). The structural proteins are located in the Nterminal portion of the polyprotein, while those associated with replication are present in the C-terminal portion (68). Among the nonstructural proteins, NS5B is an RdRp that can synthesize RNA (3, 69). Double-stranded, RNase-resistant replicative-form RNA and partially double-stranded replication intermediates have been observed early in infection in cells infected by BVDV. ...

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Section: Discussionsupporting

confidence: 80%

Template Nucleotide Moieties Required for De Novo Initiation of RNA Synthesis by a Recombinant Viral RNA-Dependent RNA Polymerase

Kim

Zhong²,

Hong³

et al. 2000

J Virol

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“…For example, recognition of the signal might occur only in double-stranded DNA, either due to base-specific recognition of the sequence in a helical context or in response to a special conformation of the DNA (which of course is also sequence-dependent). In this regard, it is interesting to note that the disposition of the conserved class II sequence relative to the site of termination (spanning an interval 6 -15 nt upstream from the termination site) is the same as the disposition of the binding domain of the T7 promoter relative to the start site for transcription (29,30). The availability of modified PTH signals that fail to cause termination may allow the selection of mutant T7 RNAPs that can utilize these signals, thereby helping to map the region of the RNAP that is responsible for signal recognition.…”

Section: Discussionmentioning

confidence: 99%

Characterization of an Unusual, Sequence-specific Termination Signal for T7 RNA Polymerase

Kukarin

Temiakov

et al. 1998

Journal of Biological Chemistry

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We have characterized an unusual type of termination signal for T7 RNA polymerase that requires a conserved 7-base pair sequence in the DNA (ATCTGTT in the nontemplate strand). Each of the nucleotides within this sequence is critical for function, as any substitutions abolish termination. The primary site of termination occurs 7 nucleotides downstream from this sequence but is context-independent (that is, the sequence around the site of termination, and in particular the nucleotide at the site of termination, need not be conserved). Termination requires the presence of the conserved sequence and its complement in duplex DNA and is abolished or diminished if the signal is placed downstream of regions in which the non-template strand is missing or mismatched. Under the latter conditions, much of the RNA product remains associated with the template. The latter results suggest that proper resolution of the transcription bubble at its trailing edge and/or displacement of the RNA product are required for termination at this class of signal.A variety of signals have been found to modulate the process of transcript elongation. In general, these have been categorized as falling into the following three classes: pause sites, which temporarily halt the RNA polymerase (RNAP) 1 but subsequently allow resumption of transcription; termination signals, which cause release of the RNA and dissociation of the transcription complex; and arrest sites, at which the RNAP may be halted for a prolonged period but may escape by cleavage and subsequent elongation of the transcript (for review, see Refs. 1 and 2). Among the termination signals, the best characterized involve the formation of a stem-loop structure in the nascent RNA (3-5). Although there have been reports of pause, arrest, or termination signals that do not involve the formation of a structured RNA (see for example Ref. 6), these signals have been less well studied. In this work, we have characterized a sequence-specific pause/termination signal for T7 RNAP and have identified the elements that are required for its function.Two types of signals are known to cause pausing and/or termination by T7 RNAP (7,8). Class I terminators, typified by the signal that is present in the late region of T7 DNA (T⌽), encode RNAs that have the potential to form stable stem-loop structures followed by a run of U residues. These features are reminiscent of many intrinsic terminators utilized by Escherichia coli RNA polymerase, and a number of bacterial termination signals have been shown to cause T7 RNAP to terminate (8 -13). Although the members of this class encode RNAs that share a typical secondary structure, they exhibit little sequence homology.A second type of termination signal recognized by T7 RNAP was first identified in the cloned human prepro-parathyroid hormone (PTH) gene (8,14). These signals (class II signals) do not encode RNAs with an apparent consistent secondary structure but share a common sequence (ATCTGTT, in the nontemplate strand (8,15,16); this work). Additional membe...

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“…It has been postulated that the stacking interactions of the Ϫ1 T in the template with Trp 422 and the ϩ1 base play a role in positioning the melted template strand within the T7 RNAP active site for correct initiation at the ϩ1-position (24,32,33). It also appears that the interactions of T7 RNAP with the upstream promoter binding region are important in the correct positioning of the template strand (29).…”

Section: Effect Of the Initiating Nucleotides On Promoter Melting-mentioning

confidence: 99%

The +2 NTP Binding Drives Open Complex Formation in T7 RNA Polymerase

Stano

Levin

Patel

2002

Journal of Biological Chemistry

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The efficiency of transcription is regulated to produce the required amount of RNA in the cell, and this can occur in numerous ways. Transcriptional efficiency is intrinsically regulated by the promoter DNA sequence during initiation through complex protein-DNA interactions. In addition, several extrinsic factors such as accessory proteins or ligands that bind to the RNAP 1 -DNA complex regulate transcription by allosteric mechanisms. The efficiency of transcription is also regulated by the concentration of substrate NTPs when the cellular NTP concentration is close to the K d of NTPs. This is found in Escherichia coli RNAP-rRNA promoter complexes, such as the rrnB P1 promoters, that have relatively weak affinity for initiating NTP, and thus rRNA synthesis is controlled by the initiating NTP concentration that varies with the cellular growth rate (1). The bacteriophage T7 RNAP is similar in that it also has a relatively weak affinity for initiating NTPs; hence, transcription is regulated by the initiating NTP concentration (2-4). It is interesting that RNAP-promoter complexes with the same initiation start sequence but different promoter core sequences respond differently to initiating NTP concentrations, because they show different affinities for initiating NTPs (3, 5). In these promoters, the template sequence at the initiation site is the same, and thus the interactions of the incoming NTP with the template and RNAP are expected to be the same; thus, it is not obvious how the core sequence regulates the affinity of initiating NTP.Both rrn P1 and T7 promoters show a common feature, and that is an unfavorable equilibrium constant for closed to open complex formation in the absence of initiating NTP. The observed K d of the initiating NTP is dependent on the efficiency of steps preceding NTP binding, such as promoter binding and opening; hence, these steps can potentially modulate the K d of the initiating NTP. Kinetic studies of T7 RNAP have shown that the observed rate of promoter opening is fast (Ͼ100 s Ϫ1 ) in the absence of initiating NTP (6 -8). However, both kinetic and structural studies suggest that the equilibrium constant of the promoter opening step is unfavorable. Fluorescence studies indicate that open complex formation is incomplete even at RNAP and DNA concentrations much above the K d of the T7 RNAP-DNA complex (8). Similarly, much of the Ϫ4 to Ϫ1 TATA region of the T7 dsDNA promoter that melts in the open complex cannot be modified by KMnO 4 , unless initiating nucleotides are present (9 -11).Incomplete conversion of closed to open complex is also observed in the rrn P1 promoters of E. coli RNAP. A closed complex between E. coli RNAP with rrn P1 promoter is formed readily, but it is not stoichiometrically converted to the open complex in the absence of initiating nucleotides (1,12,13). The presence of ATP and CTP, the initiating nucleotides, is required to both make the complex heparin-resistant and open the Ϫ10 dsDNA region (1,12,13). The instability of the open complex may be the reason for t...

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Positioning of the start site in the initiation of transcription by bacteriophage T7 RNA polymerase 1 1Edited by R. Ebright

Cited by 25 publications

References 31 publications

Template Nucleotide Moieties Required for De Novo Initiation of RNA Synthesis by a Recombinant Viral RNA-Dependent RNA Polymerase

Template Nucleotide Moieties Required for De Novo Initiation of RNA Synthesis by a Recombinant Viral RNA-Dependent RNA Polymerase

Characterization of an Unusual, Sequence-specific Termination Signal for T7 RNA Polymerase

The +2 NTP Binding Drives Open Complex Formation in T7 RNA Polymerase

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