Major Conformational Changes Occur during the Transition from an Initiation Complex to an Elongation Complex by T7 RNA Polymerase
Journal of Biological Chemistry
To examine changes that occur during the transition from an initiation complex (IC) to an elongation complex (EC) in T7 RNA polymerase (RNAP), we used nucleic acid-protein cross-linking methods to probe interactions of the RNAP with RNA and DNA in a halted EC. As the RNA is displaced from the RNA-DNA hybrid ϳ9 bp upstream from the active site (at ؊9) it interacts with a region within the specificity loop (residues 744 -750) and is directed toward a positively charged surface that surrounds
... ues Lys-302 and Lys-303. Surprisingly, the template and non-template strands of the DNA at the upstream edge of the hybrid (near the site where the RNA is displaced) interact with a region in the N-terminal domain of the RNAP (residues 172-191) that is far away from the specificity loop before isomerization (in the IC). To bring these two regions of the RNAP into proximity, major conformational changes must occur during the transition from an IC to an EC. The observed nucleic acid-protein interactions help to explain the behavior of a number of mutant RNAPs that are affected at various stages in the initiation process and in termination. Like all RNA polymerases (RNAPs), 1 T7 RNAP forms an unstable initiation complex (IC) that synthesizes and releases short abortive products before clearing the promoter and forming a stable elongation complex (EC) (Ref. 1; for review, see Ref. 2). The transition is accompanied by release of promoter contacts, changes in the size of the footprint of the polymerase on the DNA, and changes in accessibility to cleavage by a variety of proteases (3-6). Taken together, these changes indicate that significant alterations in the organization of the complex (and possibly in the structure of the RNAP) occur during the transition.