E. coli RNA Polymerase
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RNA in all cellular organisms is synthesized by a complex molecular machine, the DNA-dependent RNAP. In its simplest bacterial form, the enzyme comprises four subunits with a total molecular mass of ~400 kDa. We focus on highly characterized prokaryotic RNAPs, which share basic structure and catalytic function with more complex archaeal and eukaryotic enzymes but are controlled by a much simpler set of regulatory factors (1).
The basic elements of the transcription cycle, initiation, elongation, and termination, were elucidated through study of prokaryotes. The RNAP catalytic core, along with initiation factors (σ factors in bacteria, which combine with core to generate holoenzyme (2)), locates promoter sequences within the duplex DNA, forms the open promoter complex (RPo) by unwinding the DNA surrounding the transcription start site, initiates synthesis of an RNA chain, elongates the RNA processively in an elongation complex (EC) while translocating itself and the transcription bubble along the DNA template, and finally releases itself and the completed transcript from the DNA when termination signals are encountered. |
The transcription cycle is marked by a series of stable RNAP complexes (core (3) → holo(4) →RPo (5) → EC (6) , all structures determined by the Darst lab using X-ray crystallography or cryo-electron microscopy) that interconvert through transient intermediates involving large conformational changes in the nucleic acids, the RNAP, or both. At every stage of the transcription cycle, RNAP function is modulated by interactions with hundreds of extrinsic regulatory factors (7) . Moreover, bacteriophage have evolved extrinsic factors that use ingenious mechanisms to subvert the host transcription process for their own purposes (8).
A detailed structural and functional understanding of the entire transcription cycle is essential to explain the fundamental control of gene expression and to target RNAP with small-molecule antibiotics (9). Moreover, a complete understanding of how a complex, molecular machine uses binding and chemical energy to effect conformational changes that drive the cycle, and how regulators modulate the cycle, is of fundamental interest. We continue to use a combination of X-ray crystallography, cryo-electron microscopy, and other biophysical and biochemical approaches to fill the gaps in our understanding of the bacterial transcription cycle, particularly large regulatory complexes and unstable transition states between the stable states of the cycle.
A detailed structural and functional understanding of the entire transcription cycle is essential to explain the fundamental control of gene expression and to target RNAP with small-molecule antibiotics (9). Moreover, a complete understanding of how a complex, molecular machine uses binding and chemical energy to effect conformational changes that drive the cycle, and how regulators modulate the cycle, is of fundamental interest. We continue to use a combination of X-ray crystallography, cryo-electron microscopy, and other biophysical and biochemical approaches to fill the gaps in our understanding of the bacterial transcription cycle, particularly large regulatory complexes and unstable transition states between the stable states of the cycle.