Structural basis for transcription elongation by bacterial RNA polymerase

The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-A resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the beta'-subunit 'lid' loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination.     

RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals

The large subunit of RNA polymerase II contains a highly conserved and essential heptapeptide repeat (Pro-Thr-Ser-Pro-Ser-Tyr-Ser) at its carboxy terminus. Saccharomyces cerevisiae cells are inviable if their RNA polymerase II large subunit genes encode fewer than 10 complete heptapeptide repeats; if they encode 10 to 12 complete repeats cells are temperature-sensitive and cold-sensitive, but 13 or more complete repeats will allow wild-type growth at all temperatures. Cells containing C-terminal domains (CTDs) of 10 to 12 complete repeats are also inositol auxotrophs. The phenotypes associated with these CTD mutations are not a consequence of an instability of the large subunit; rather, they seem to reflect a functional deficiency of the mutant enzyme. We show here that partial deletion mutations in RNA polymerase II CTD affect the ability of the enzyme to respond to signals from upstream activating sequences in a subset of promoters in yeast. The number of heptapeptide repeats required for maximal response to signals from these sequences differs from one upstream activating sequence to another. One of the upstream elements that is sensitive to truncations of the CTD is the 17-base-pair site bound by the GAL4 transactivating factor.