Nucleic Acids Research, 1993, Vol. 21, No. 8 1927-1933
© 1993
MOLECULAR BIOLOGY |
Transcription by SP6 RNA polymerase exhibits an ATP dependence that is influenced by promoter topology
Cold Spring Harbor Laboratory PO Box 100, Cold Spring Harbor, NY 11724, USA
* To whom correspondence should be addressed
Received December 9, 1992. Revised March 10, 1993. Accepted March 10, 1993.
Transcription of linearized DNA templates by SP6 RNA polymerase requires a higher concentration of ATP than of the other three nucleotides. This requirement is not shared by T7 RNA polymerase. The ATP requirement is partially relieved when the SP6 template Is supercolled but not when It Is relaxed circular DNA. The effect of supercolllng Is eliminated by replacement of the A·T rich sequence downstream from the SP6 promoter with a G·C rich sequence. Examination of the reaction products Indicates that the ATP dependence of transcription from a linear template is not due to an ATPase activity or to the premature termination of transcription at low ATP concentration. These data suggest that the initiation of transcription by SP6 RNA polymerase requires partial denaturation of the template in the promoter-proximal region, and that this requirement can be satisfied by negative supercolllng or by increasing the ATP concentration. ATP also reduces, but does not eliminate, the abortive transcription that leads to the production of short, prematurely terminated transcripts by SP6 polymerase from supercolled templates.