Abstract
Termination of transcription of vaccinia virus early genes requires the virion form of the viral RNA polymerase (RNAP), a termination signal (UUUUUNU) in the nascent RNA, vaccinia termination factor, nucleoside triphosphate phosphohydrolase I (NPH I), and ATP. NPH I uses ATP hydrolysis to mediate transcript release, and in vitro, ATPase activity requires single-stranded DNA. NPH I shows sequence similarity with the DEXH-box family of proteins, which includes an Escherichia coli ATP-dependent motor protein, Mfd. Mfd releases transcripts and rescues arrested transcription complexes by moving the transcription elongation complex downstream on the DNA template in the absence of transcription elongation. This mechanism is known as forward translocation. In this study, we demonstrate that NPH I also uses forward translocation to catalyze transcript release from viral RNAP. Moreover, we show that NPH I-mediated release can occur at a stalled RNAP in the absence of vaccinia termination factor and U(5)NU when transcription elongation is prevented.
Highlights
The enzyme nucleoside triphosphate phosphohydrolase I (NPH I) is involved in vaccinia early gene transcription termination
We demonstrate that NPH I uses forward translocation to catalyze transcript release from viral RNA polymerase (RNAP)
We show that NPH I-mediated transcript release can occur independent of vaccinia termination factor (VTF) and U5NU when NPH I is stimulated with dATP, as opposed to ATP
Summary
The enzyme NPH I is involved in vaccinia early gene transcription termination. Results: Transcript release occurred in the presence of NPH I and dATP only when RNA polymerase can move forward in the absence of transcription elongation. Conclusion: NPH I causes transcript release during vaccinia early gene transcription termination using the mechanism of forward translocation. NPH I shows sequence similarity with the DEXH-box family of proteins, which includes an Escherichia coli ATP-dependent motor protein, Mfd. Mfd releases transcripts and rescues arrested transcription complexes by moving the transcription elongation complex downstream on the DNA template in the absence of transcription elongation. Mfd releases transcripts and rescues arrested transcription complexes by moving the transcription elongation complex downstream on the DNA template in the absence of transcription elongation This mechanism is known as forward translocation. We show that NPH I-mediated release can occur at a stalled RNAP in the absence of vaccinia termination factor and U5NU when transcription elongation is prevented
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