New Views of RNA Polymerase

The reports, which appear in the March 2002 issue of Molecular Cell and the May 17, 2002, issue of Science, illuminate how the process of transcription begins in bacteria and provide clues for future development of new antibacterial drugs.

RNA polymerases are among the largest and most complex proteins of living organisms. These holoenzymes are similar in overall structure in both prokaryotes and eukaryotes; each polymerase consists of two large polypeptide subunits and about six to 10 smaller ones. Typically the two largest polypeptides and a few of the smaller polypeptides form a core enzyme that is capable of transcribing essentially any DNA sequence, natural or artificial, into an RNA copy. The remaining smaller polypeptides restrict the transcribing capabilities of the enzyme to natural DNAs containing a promoter. The holoenzyme's sigma subunit ‘tells' RNAP where in the genomic DNA to start transcription. It does this by recognizing the promoter region and ‘melting' the DNA to expose one of the double-helical strands the RNAP will use as the template to synthesize RNA.

Investigators from Rockefeller University (New York NY, USA) used X-ray crystallography to study RNAP from the thermophilic bacterium Thermus aquaticus. As the crystals of bacterial RNAP produced did not allow high-resolution structural analysis, they developed an alternative hybrid approach. "From previous and parallel work in our lab, we had high resolution structures of the RNA polymerase and parts of the sigma subunit,” explained Dr. Seth Darst. "We fitted the high resolution structures into the lower resolution structure to obtain the structure of the holoenzyme. And since the structure of DNA is known, we used that to solve the structure of the holoenzyme bound to the promoter fragment.”

It is now clear that the sigma subunit contains four protein domains connected to one another by short stretches of amino acids called "linkers.” The domains of the sigma subunit spread out and bind to one face of the core enzyme, which positions sigma to bind to DNA. One of the linkers, containing about 33 amino acids, is much longer than the others. This linker goes inside the polymerase towards the active site and back out, producing a very unusual protein-protein interaction.
"These sigma domains sit on the surface of the RNA polymerase, but this very long linker is buried inside the polymerase, near the active site of the enzyme,” says Dr. Darst. "In order to establish this complex, there needs to be a lot of conformational changes to get the linker inside. The linker sticks inside the polymerase and actually blocks the path of the RNA. As the RNA transcript begins to be elongated, it has to push this linker out from a channel inside the polymerase. The linker's not completely pushed out until the RNA becomes about 12 nucleotides long.”

A transition occurs when the RNA transcript reaches a length of 12 nucleotides: the complex becomes much more stable and the elongation phase begins. "We think that signal to go into elongation occurs when the RNA becomes long enough to fill the channel,” says Darst. "It pushes the linker peptide out, and then sigma begins to fall off.”

A better understanding of transcription in bacteria could lead to new drug targets to treat bacterial infections, particularly those that are resistant to current antibiotics.



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