RNA polymerase is required in all living cells

Transcription, the process by which cells make transient RNA copies of the archival information stored in DNA, lies at the heart of cellular regulation, and understanding the complex machinery involved in the synthesis of RNA is critical to understanding that regulation. Many cancers and genetic diseases arise from improperly regulated transcription. Too much or too little of a good thing, at the wrong time, can be lethal.

As a result of its central role in biology, RNA polymerase is a key target or tool in biotechnology and in pharmaceutical development.

An RNA polymerase is a complex molecular nanomachine. In the cell, it must respond to intracellular signals and then initiate the following multi-step process:

1. Find (bind to) the unique spot along the DNA encoding a particular transcript
2. Melt open the DNA duplex at that location
3. Direct the templating strand into the enzyme active site
4. Promote binding of the first two nucleoside triphosphates, as encoded in the DNA
5. Form a bond between those two nucleotides
6. Translocate forward by one base, placing the next base in the template strand into the active site
7. Repeat - thousands of times
8. Recognize a termination sequence in the DNA
9. Terminate elongation and release the RNA product

But it gets more complicated

• Binding two nucleoside triphosphates into the active site is very different from binding a long, nascent RNA and a single nucleoside triphosphate. How can a single active site accommodate both the initial phase and the later phase?
• The initially synthesized end (5') of the RNA must at some point be displaced from the template DNA, to allow the two DNA strands to reassociate (reanneal).
• Transcribing complexes are VERY stable, EXCEPT for within the first 8-10 nucleotides. Within that region, RNA polymerase will initiate, extend for a bit, release the very short (abortive) product RNA, and then start over (abortive cycling).

We now have crystal structures for eukaryotic, prokaryotic, and phage RNA polymerases, but many questions remain regarding the mechanisms of the individual phases of transcription. Indeed, regulation occurs not only at the initial selection of the promoter, but also at the initial synthesis step, during the initial movement away from the promoter, during elongation, and at sequence-specific termination. The structure-function relationships underlying each of these processes are key to understanding transcription.

T7 RNA polymerase is the simplest and best-understood of the RNA polymerases. In a sense, it is the core of an RNA polymerase. As such, it represents an ideal model system in which to study transcription.