Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes

Hindman, Ryan; Gollnick, Paul

doi:10.1074/jbc.m116.730135

Cited by 5 publications

(3 citation statements)

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“…It has previously been demonstrated that both Rap94 and VTF/CE are involved in the recognition of the termination motif, which may pause the elongating polymerase (Christen et al, 2008;Luo et al, 1995;Tate and Gollnick, 2015). NPH-I may then cause transcript extrusion from the active site by a 5 0 to 3 0 translocase activity on the non-template strand (Hindman and Gollnick, 2016;Tate and Gollnick, 2011). Provided the observed location of the CEC near the putative RNA exit tunnel is relevant for a termination intermediate, CEC may be involved in the recognition of the termination signal.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes

Grimm

Hillen

Bedenk

et al. 2019

Cell

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Section: Discussionmentioning

confidence: 99%

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes

Grimm

Hillen

Bedenk

et al. 2019

Cell

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“…Although Rap94 and other transcription factors must be displaced from the vRNAP surface to form the CCC, they can remain flexibly associated with the polymerase in the infected cell. After 5 0 cap synthesis, transcription elongation can proceed to the end of the gene, where termination is mediated by NPH-I and VTF (CE) (Christen et al, 1999;Hindman and Gollnick, 2016). In the future, structural insights into initiation and termination should reveal how the virus-specific factors Rap94, VETF, and NPH-I mediate these phases of the transcription cycle.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes

Hillen

Bartuli

Grimm

et al. 2019

Cell

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Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate m 7 G-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of core and complete vRNAP complexes of the prototypic Vaccinia poxvirus (Grimm et al., 2019; in this issue of Cell). Here, we present the cryo-electron microscopy (cryo-EM) structures of Vaccinia vRNAP in the form of a transcribing elongation complex and in the form of a co-transcriptional capping complex that contains the viral capping enzyme (CE). The trifunctional CE forms two mobile modules that bind the polymerase surface around the RNA exit tunnel. RNA extends from the vRNAP active site through this tunnel and into the active site of the CE triphosphatase. Structural comparisons suggest that growing RNA triggers large-scale rearrangements on the surface of the transcription machinery during the transition from transcription initiation to RNA capping and elongation. Our structures unravel the basis for synthesis and co-transcriptional modification of poxvirus RNA.

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“…However, the mechanism and structural basis of termination by these factors remains opaque. Termination of early VACV genes is not reliant on A18, but by D11 (aka NPH-I) ( 83 , 84 ) or I8 (NPH-II) ( 85 – 87 ), which terminate transcription immediately following pyrimidine-rich sequences, both in the absence and presence of the upstream UUUUUNU motif ( 23 ).…”

Section: Discussionmentioning

confidence: 99%

Transcription termination and readthrough in African swine fever virus

Cackett,

Sýkora,

Portugal

et al. 2024

Front. Immunol.

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IntroductionAfrican swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that encodes its own host-like RNA polymerase (RNAP) and factors required to produce mature mRNA. The formation of accurate mRNA 3′ ends by ASFV RNAP depends on transcription termination, likely enabled by a combination of sequence motifs and transcription factors, although these are poorly understood. The termination of any RNAP is rarely 100% efficient, and the transcriptional “readthrough” at terminators can generate long mRNAs which may interfere with the expression of downstream genes. ASFV transcriptome analyses reveal a landscape of heterogeneous mRNA 3′ termini, likely a combination of bona fide termination sites and the result of mRNA degradation and processing. While short-read sequencing (SRS) like 3′ RNA-seq indicates an accumulation of mRNA 3′ ends at specific sites, it cannot inform about which promoters and transcription start sites (TSSs) directed their synthesis, i.e., information about the complete and unprocessed mRNAs at nucleotide resolution. MethodsHere, we report a rigorous analysis of full-length ASFV transcripts using long-read sequencing (LRS). We systematically compared transcription termination sites predicted from SRS 3′ RNA-seq with 3′ ends mapped by LRS during early and late infection. ResultsUsing in-vitro transcription assays, we show that recombinant ASFV RNAP terminates transcription at polyT stretches in the non-template strand, similar to the archaeal RNAP or eukaryotic RNAPIII, unaided by secondary RNA structures or predicted viral termination factors. Our results cement this T-rich motif (U-rich in the RNA) as a universal transcription termination signal in ASFV. Many genes share the usage of the same terminators, while genes can also use a range of terminators to generate transcript isoforms varying enormously in length. A key factor in the latter phenomenon is the highly abundant terminator readthrough we observed, which is more prevalent during late compared with early infection. DiscussionThis indicates that ASFV mRNAs under the control of late gene promoters utilize different termination mechanisms and factors to early promoters and/or that cellular factors influence the viral transcriptome landscape differently during the late stages of infection.

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Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes

Cited by 5 publications

References 61 publications

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes

Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes

Transcription termination and readthrough in African swine fever virus

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