Left-handed orientation of histidine-tagged RNA polymerase complexes imaged by atomic force microscopy

Atomic force microscopy of histidine-tagged Escherichia coli RNA polymerase (RNAP) complexes has shown that, for complexes that are asymmetric because the DNA ends are of different lengths, over 90% of the complexes bind to mica in a left-handed orientation. DNA usually appears to bind to the side of RNAP in AFM images, as opposed to passing through the middle of the RNAP. This is consistent with the off-center location of the DNA-binding groove in E. coli RNAP and other polymerases. Complexes with wild-type RNAP bind to mica in both left-handed and right-handed orientations. These AFM results are consistent with a model in which the downstream end of the DNA lies on the lower projection of RNAP, when the RNAP is oriented such that its projections or fingers are on the left side of the molecule. AFM images can differentiate between elongation complexes containing RNA transcripts of 13-34 bases and elongation complexes containing RNA transcripts of 52-64 bases, because RNAP is more nearly centered on the DNA in the 13- to 34-base complexes than in the 52- to 64-base complexes.