Abstract
RNA polymerase II (RNAPII) undergoes structural changes during the transitions from initiation, elongation, and termination, which are aided by a collection of proteins called elongation factors. NusG/Spt5 is the only elongation factor conserved in all domains of life. Although much information exists about the interactions between NusG/Spt5 and RNA polymerase in prokaryotes, little is known about how the binding of eukaryotic Spt4/5 affects the biochemical activities of RNAPII. We characterized the activities of Spt4/5 and interrogated the structural features of Spt5 required for it to interact with elongation complexes, bind nucleic acids, and promote transcription elongation. The eukaryotic specific regions of Spt5 containing the Kyrpides, Ouzounis, Woese domains are involved in stabilizing the association with the RNAPII elongation complex, which also requires the presence of the nascent transcript. Interestingly, we identify a region within the conserved NusG N-terminal (NGN) domain of Spt5 that contacts the non-template strand of DNA both upstream of RNAPII and in the transcription bubble. Mutating charged residues in this region of Spt5 did not prevent Spt4/5 binding to elongation complexes, but abrogated the cross-linking of Spt5 to DNA and the anti-arrest properties of Spt4/5, thus suggesting that contact between Spt5 (NGN) and DNA is required for Spt4/5 to promote elongation. We propose that the mechanism of how Spt5/NGN promotes elongation is fundamentally conserved; however, the eukaryotic specific regions of the protein evolved so that it can serve as a platform for other elongation factors and maintain its association with RNAPII as it navigates genomes packaged into chromatin.
Highlights
The conversion of DNA to RNA is a fundamental aspect of all life, and this process is carried out by RNA polymerases (RNAPs).2 These enzymatic powerhouses must maintain both
RNA polymerase II (RNAPII) elongation was initiated from a 3Ј single strand extension on a duplex template to assemble an Elongation complexes (ECs) with a defined transcript produced from a G-less cassette (Fig. 1A)
Changes occur to RNAPII as it transitions from the pre-initiation complex into a productive elongation complex
Summary
The conversion of DNA to RNA is a fundamental aspect of all life, and this process is carried out by RNA polymerases (RNAPs). These enzymatic powerhouses must maintain both. The movement of the trigger loop and bridge helix is linked to the formation of RNA hairpins, which regulate RNAP pausing [13, 16, 17] This method of pausing is not known to exist in eukaryotes, x-ray crystal structures of yeast RNAPII in different stages of elongation have generated a model in which the movement of the bridge helix and trigger loop can be coupled to translocation through the non-template strand of DNA [18]. This information implies that the nucleic acid scaffold is critical in maintaining active RNAP during elongation. “Bridging” of the two lobes of RNAP occurs through the universally conserved NGN domain (NusG N-terminal region), and all known biomechanical properties of Spt are linked to this domain [1, 7, 19, 20]
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