A cinematographic view of Escherichia coli RNA polymerase translocation

Abstract

A series of RNA synthesizing transcription complexes, initiated at the T7 A1 promoter and halted at specific base positions ranging from +12 to +40, were analyzed by footprinting techniques; exonuclease III was used to determine the position of the bound RNA polymerase on the DNA and hydroxyl radicals were used to visualize the protein-DNA contact sites within the protected areas. In the binding (open) complex without RNA there are two DNA-domains, differing in their protection pattern. The first, extending from position +18 to -13, termed 'melting domain', is fully protected, whereas the second, extending from -14 to -55, termed 'recognition domain', shows only partial protection. At this domain, RNA polymerase is attached to one side of the DNA only, as indicated by the 10-bp periodicity of the protection pattern. Our data show that the formation of a mature RNA transcribing complex is characterized by dissociation of the RNA polymerase from the recognition domain, whereby the size of the melting domain remains constant. This process is accomplished if the nascent RNA has reached a length of 11 bases. As the RNA reaches a length of 20 bases, the size of the melting domain decreases from ~30 to 23 bp. Further RNA synthesis leaves the protection pattern essentially unchanged. These data demonstrate that the formation of a mature RNA transcribing complex can be described by at least two transitions.