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

Ryan Hindman,Paul Gollnick

doi:10.1074/jbc.m116.730135

Abstract

Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5' to 3' translocase on single-stranded DNA.

Highlights

Vaccinia virus is a member of the poxvirus family, which are large double-stranded DNA viruses that conduct their entire infective cycle within the cytoplasm of the infected cell
This signal is required for termination of ongoing transcription [11]; it is not required for nucleoside triphosphate phosphohydrolase I (NPH I)-mediated transcript release from a stalled transcription elongation complex (TEC) [11, 15]
Transcription termination involves the cessation of RNA polymerization and release of nascent RNA from the transcription complex

Summary

Introduction

Vaccinia virus is a member of the poxvirus family, which are large double-stranded DNA viruses that conduct their entire infective cycle within the cytoplasm of the infected cell. For oligo-based transcription templates that lacked non-template DNA or contained mismatches, the sequence of the completed template strand was identical to Ter29 [18] except that the termination signal was moved downstream to ϩ46, and the sequence following the end of the A-less cassette was shortened. Adding an antisense oligonucleotide complementary to 26 residues at the 3Ј end of the nascent transcript in the second step of the assay resulted in release of ϳ50% of the transcripts from the fully double-stranded template NT90 (Fig. 2, D, Lanes 4 and 5, and E, NT90).

Results

Conclusion