Transcription bubble, structure

Figure 28-4 (A) Hypothetical structure of a "transcription bubble" formed by an RNA polymerase. Shown is a double-stranded length of DNA with the unwound bubble in the center. This contains a short DNA-RNA hybrid helix formed by the growing mRNA. The DNA double helix is undergoing separation at point A as is the hybrid helix at point B. NTP is the ribonucleotide triphosphate substrate. See Yager and von Hippel.71 (B) Stereoscopic view of the structure of RNA polymerase from Thermus aquaticus in a complex with a promoter DNA. Included are the al, all, (0, (3, P, and a subunits. However, the a C-terminal domains have been omitted. The template (t) strand passes through a tunnel, which is formed by the P and P subunits and two of the structural domains of the a subunit. The nontemplate (nt) strand follows a different path. The position of the -10, -35, and UP elements of the DNA are marked. From Murakami et al.33d Courtesy of Seth A. Darst.

How does this combination hairpin-oligo(U) structure terminate transcription First, it seems likely that RNA polymerase pauses immediately after it has synthesized a stretch of RNA that folds into a hairpin. Furthermore, the RNA-DNA hybrid helix produced after the hairpin is unstable because its rU-dA base pairs are the weakest of the four kinds. Hence, the pause in transcription caused by the hairpin permits the weakly bound nascent RNA to dissociate from the DNA template and then from the enzyme. The solitary DNA template strand rejoins its partner to re-form the DNA duplex, and the transcription bubble closes. 

Because the Pol II core alone is sufficient to maintain the transcription bubble and the DNA-RNA hybrid during RNA chain elongation, there must be exposed elements on the enzyme surface that keep the nucleic acid strands apart. Protein elements are needed to separate the DNA strands downstream of the active site and to separate the RNA from the DNA template strand at the upstream end of the hybrid. On the basis of their location with respect to nucleic acids, several Pol II structural elements are predicted to maintain the bubble and the hybrid. These proposals are currently tested by site-directed mutagenesis. Separation of the DNA strands at the downstream edge of the bubble may be attributed to binding of the DNA template strand by switch regions 1 and 2 and to blocking of the path of the nontemplate strand by fork loop 2. In the Pol II-TFIIS complex structure, fork loop 2 is ordered and restricts the cleft to a diameter of 15 A, consistent with the proposal that this loop removes the DNA nontemplate strand from the template strand before the active site. 

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