We have characterized elongation complexes (ECs) of RNA polymerase from the extremely thermophilic bacterium, Thermus thermophilus. We found that complexes assembled on nucleic acid scaffolds are transcriptionally competent at high temperature (50 - 80°C) and, depending upon the organization of the scaffold, possess distinct translocation conformations. ECs assembled on scaffolds with a 9 bp RNA:DNA hybrid are highly stable, resistant to pyrophosphorolysis, and are in the posttranslocated state. ECs with an RNA:DNA hybrid longer or shorter than 9 bp appear to be in a pretranslocated state, as evidenced by their sensitivity to pyrophosphorolysis, GreA-induced cleavage, and exonuclease footprinting. Both pretranslocated (8 bp RNA:DNA hybrid) and posttranslocated (9 bp RNA:DNA hybrid) complexes were crystallized in distinct crystal forms, supporting the homogeneity of the conformational states in these complexes. Crystals of a posttranslocated complex were used to collect diffraction data at atomic resolution. © 2006 Oxford University Press.