Drosophila factor 2, an RNA polymerase II transcript release factor, has DNA-dependent ATPase activity

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Drosophila factor 2 is a component of negative transcription elongation factor (N-TEF).
• N-TEF releases RNA polymerase II transcripts in an ATP-dependent manner.

Purpose of the Study:

• To investigate the specific nucleotide requirements for Drosophila factor 2's transcript release activity.
• To characterize the ATPase activity of Drosophila factor 2 and its correlation with transcript release.
• To determine if additional protein cofactors are necessary for factor 2's function.

Main Methods:

• Assessing transcript release activity using RNA polymerase II elongation complexes on a dC-tailed template.
• Measuring DNA-dependent ATPase activity of factor 2.
• Evaluating the effect of various nucleotide analogs (ATPgammaS, AMP-PNP) and NTPs on factor 2 activity.

Main Results:

• Factor 2's transcript release activity strictly requires ATP or dATP; other NTPs and analogs do not support this function.
• Factor 2 exhibits significant DNA-dependent ATPase activity that directly correlates with its transcript release capability.
• The ATPase activity was characterized with an apparent Km(ATP) of 28 microM and Kcat of 140 min-1 at 20 microg/ml DNA.
• No other protein cofactors are needed for factor 2-mediated transcript release.
• Template renaturation is necessary for factor 2 to function in the dC-tailed template assay.

Conclusions:

• Drosophila factor 2 possesses intrinsic transcript release activity dependent on ATP hydrolysis.
• The DNA-dependent ATPase activity is central to the transcript release mechanism mediated by factor 2.
• Factor 2 functions independently of other cofactors, highlighting its direct role in transcription regulation.