Proposal for RNAP Transcription Complex Stability

Numerous X-ray diffraction studies have attempted to depict accurate views of a transcribing DNA dependent- RNA polymerase. Watson-Crick interactions occur between transient DNA-RNA hybrids constructed from template-dependent RNA synthesis (Severinov 2001). Despite this, uncertainty remains about the required molecular components involved in the processivity and stability of the RNA polymerase transcription complex (Severinov 2001). Evidence suggests that the unphosphorylated RNAP IIa subunit of Rbp 1 initially binds to a promoter, and the phosphorylated RNAP IIo subunit carries out elongation (Weaver 2002). During formation of the promoter-complex, RNAPs produce short, abortive nascent RNA oligomers ranging from 2-8 nucleotides long (Severinov 2001). When an RNA oligomer of 9 bases is created, it is stably associated with the polymerase transcription complex, and the RNAP clears the promoter region to processively elongate the chain (Severinov 2001). Once RNAP encounters an RNA hairpin followed by a run of uridines, the polymerase pauses and releases nucleic acids because the stability of the transcription complex is altered (Severinov 2001).

Image of yeast RNA polymerase II (Weaver 2002, p276)

A 25-Ǻ channel goes through RNAP with mainly Rpb1 on one side and Rpb2 on the other (Weaver 2002). The opening of the channel is composed of Rpb1, Rpb9, and the Rpb5 subunit, which acts to grasp DNA (Weaver 2002). Studies by Roger Kornberg and colleagues show that DNA and RNA bind in the central cleft between Rpb1 and Rpb2 subunits. The surface charge distribution of yeast at 2.8 Ǻ resolution shows that hundreds of positively charged amino acid side chains line the cleft appropriately for interaction with negatively charged DNA and RNA (Kornberg 2001). Duplex DNA enters RNAP II and unwinds 3 bases before the active site. The template strand makes a 90˚ bend and points downward toward the floor of the cleft for readout at the active site (Kornberg 2001). The ribonucleotide on the RNA chain is then base paired to the DNA strand forming a RNA-DNA hybrid (Kornberg 2001). The structure determination of a crystallized transcribing RNAP proved that the RNA and DNA interact in the RNAP cleft (Kornberg 2001).

Elongation complex
Adopted from http://www.sciencemag.org/cgi/content/full/288/5466/640/F6

The Rpb2 subunit of RNAP forms a wall on the upstream side of the active site that deters DNA from extending upstream with an orientation like downstream DNA (Weaver 2002). The RNA-DNA hybrid upstream from the active site is at an angle (dashed line) to the downstream DNA (Weaver 2002). The pores depicted allow exit of the growing RNA oligomer and hold a sliding clamp (composed of Rpb1, Rpb2, and Rpb6 subunits) on the DNA improving processivity (Weaver 2002).

DNA-RNA hybrid model of transcription complex (Severinov 2001,p6)

Several models of the mechanism of RNAP transcription have focused on the length and role of the RNA-DNA hybrid. It is hypothesized that the stabilization of RNA in the transcription bubble results from protein-RNA contacts or the establishment of a full-length RNA-DNA hybrid (Severinov 2001). A hybrid consisting of less than 3 base pairs that remains throughout elongation implies that stability of the transcription complex is due to the strength of protein-DNA interactions (Severinov 2001). However, a hybrid 8-9 base pairs long would explain the stabilization of the nascent RNA in the transcription complex during promoter clearance (Severinov 2001). This hybrid length would also confer that the relative instabilities of the transcription initiation and termination complexes are due to short hybrid lengths (Severinov 2001).

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