Abstract
Drosophila factor 2, an RNA polymerase II transcript release factor, exhibits a DNA-dependent ATPase activity (Xie, Z., and Price D. H. (1997) J. Biol. Chem. 272, 31902-31907). We examined the nucleic acid requirement and found that only double-stranded DNA (dsDNA) effectively activated the ATPase. Single-stranded DNA (ssDNA) not only failed to activate the ATPase, but suppressed the dsDNA-dependent ATPase. Gel mobility shift assays showed that factor 2 formed stable complexes with dsDNA or ssDNA in the absence of ATP. However, in the presence of ATP, the interaction of factor 2 with dsDNA was destabilized, while the ssDNA-factor 2 complexes were not affected. The interaction of factor 2 with dsDNA was sensitive to increasing salt concentrations and was competed by ssDNA. In both cases, loss of binding of factor 2 to dsDNA was mirrored by a decrease in ATPase and transcript release activity, suggesting that the interaction of factor 2 with dsDNA is important in coupling the ATPase with the transcript release activity. Although the properties of factor 2 suggested that it might have helicase activity, we were unable to detect any DNA unwinding activity associated with factor 2.
Highlights
All information gathered on factor 2 function suggests that ATP hydrolysis is a key reaction for the transcript release activity of factor 2, but how it associates with elongation complexes and how the ATP hydrolysis is coupled to the transcript release event is not clear
Factor 2 Has No Apparent DNA Helicase Activity—Since factor 2 was able to bind double-stranded DNA (dsDNA) and hydrolyze ATP, we investigated whether the factor was able to utilize the energy from ATP hydrolysis to unwind duplex DNA
DsDNA effectively supported the ATPase activity, with supercoiled DNA having a slightly higher efficiency compared with linearized DNA. Single-stranded DNA (ssDNA) failed to activate but suppressed the dsDNA-dependent ATPase activity of factor 2
Summary
N-TEF, negative transcription elongation factors; dsDNA, double-stranded DNA; ssDNA, single-strand DNA; P-TEF, positive transcription elongation factors; HIV, human immunodeficiency virus; TRCF, transcription repair coupling factor; bp, base pairs(s); ATP␥S, adenosine 5Ј-O-(thiotriphosphate). Escherichia coli termination factor rho has an RNA-dependent ATPase activity that allows the translocation of rho along nascent transcripts to track elongation complexes and unwind RNA-DNA hybrids in transcription bubbles [15,16,17]. TRCF interacts with DNA and may associate with elongation complexes through its interaction with DNA and RNA polymerase and cause the release of RNA polymerase and nascent transcripts by hydrolyzing ATP [20, 21] Though it is not clear how factor 2 associates with elongation complexes, the finding that dsDNA is able to activate the ATPase activity of factor 2 [14] and the observation that factor 2 is able to bind DNA affinity column suggest that factor 2 may bind DNA and associate with elongation complexes through the interaction with DNA template. We explored the potential helicase activity of factor 2
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