A Structural Model of Transcription Elongation

Abstract

The path of the nucleic acids through a transcription elongation complex was tracked by mapping cross-links between bacterial RNA polymerase (RNAP) and transcript RNA or template DNA onto the x-ray crystal structure. In the resulting model, the downstream duplex DNA is nestled in a trough formed by the beta' subunit and enclosed on top by the beta subunit. In the RNAP channel, the RNA/DNA hybrid extends from the enzyme active site, along a region of the beta subunit harboring rifampicin resistance mutations, to the beta' subunit "rudder." The single-stranded RNA is then extruded through another channel formed by the beta-subunit flap domain. The model provides insight into the functional properties of the transcription complex.

Thermodynamic and kinetic modeling of transcriptional pausing

“…The transcription process is carried out by RNA polymerase (RNAP) (1-3), a multisubunit molecular motor, the basic structure of which is conserved from bacteria to eukaryotes (4). Over the past decade a great deal has been learned about the structure of RNAP, particularly in the context of yeast (5,6), thermophilic bacteria (7,8), and Escherichia coli (9)(10)(11)(12). Given the high degree of structural conservation of RNAP, and the synthesis of structural, biochemical, and kinetic information from bacteria and yeast, we are able to begin building mechanistic models of transcription valid across species.…”

Single-Molecule Studies of RNA Polymerase: Motoring Along

“…Prior to determination of the crystal structure of RNAP (74,75) and to the elucidation of the paths taken by nucleic acids through the elongating enzyme (73), AFM images of TECs correctly measured the large bend angle between the upstream and downstream DNA, which is close to 90° (48). Longer RNA transcripts could occasionally be visualized in TEC images, as well (76).…”

“…For the case of intrinsic termination, the force generated by a terminator hairpin during folding is not thought to be sufficient to release RNA at most sites along the DNA (11). Therefore, the hybrid-destabilizing effect of the U-rich tract, possibly aided by other mechanisms, such as hairpin stem invasion (73), allostery (128), or forward translocation (131) must play some role in the energetics of intrinsic termination. Future singlemolecule experiments should be able to probe these energetics.…”

“…However, the forces that can be applied to the nascent RNA without impairing transcription vastly exceed the forces required to unzip or shear apart an 8-9-bp RNA:DNA duplex. A "sliding clamp" model, where extensive protein-nucleic acid contacts within the polymerase contribute significantly to RNA retention has been proposed to explain the overall TEC stability (73). Such a clamp, consisting of a narrow protein channel surrounding the hybrid, would also prevent any significant shearing motions between the RNA and DNA strands under load, because confinement inside a channel would lead to significant steric clashes between bases (11).…”

T7 RNA polymerase transcription complex: What you see is not what you get

“…The upstream portion of the hybrid contributes the most to transcription complex stability (8,9). Furthermore, a structural model of bacterial RNA Pelongation complex built by superimposing the positions of protein-nucleic acid crosslinks onto a high-resolution structure neatly accommodates an 8-bp hybrid (10). Despite a lack of sequence similarity between the two RNAP families, the essential elements of the transcription cycle seem to be conserved (11).…”