Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest

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.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 high levels of fidelity and processivity over long distances to ensure that RNAs are accurately produced on a time scale amenable to life. Families of proteins called elongation factors have evolved to assist RNA polymerases during transcription elongation. The oldest and most conserved of these factors is the NusG/suppressor of Ty element (Spt) 5 family (1, 2). NusG is the eubacterial version of Spt5 and functions as a single polypeptide; however, archaea and eukaryotic Spt5 associate with an additional small protein, Spt4. In yeast, SPT5 is essential, but SPT4 is not. Deleting the gene encoding Spt4 impairs elongation, transcription-coupled repair, and mRNA processing (2-5). Some of the functions of Spt4 may be partially dependent on its ability to prevent degradation of Spt5 in cells (4). The NusG/Spt5 family of proteins has been shown to enhance RNA polymerase transcription elongation in all domains of life (6 -10). NusG regulates RNAP activity by stabilizing the post-translocated state thereby inhibiting backtracking and reducing long lifetime pauses (6, 11). The NusG homolog RfaH has also been implicated in regulating movement of the RNAP bridge hel...

NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble

“…The stimulation of RNA chain elongation by NusG has been documented in vitro for bacterial (Burova et al, 1995), archaeal (Hirtreiter et al, 2010) and eukaryotic (Wada et al, 1998) transcription systems. The NTD is sufficient for these effects on elongation, but the structural elements that superimpose well between the bacterial NusG NTD and the archaeal Spt5 NTD (Figure 8C) are limited to ( i ) the beta sheet that comprises the RNAP-binding site, ( ii ) the conserved five amino acid loop that we implicate in the anti-backtracking action of E. coli NusG, and ( iii ) the N-terminus of the α-helix that follows this loop and possibly interacts with the single-stranded non-template DNA (Crickard et al, 2016). The lack of the strong conservation of the surface residues (Figure 8D) suggests that the anti-backtracking activity may be determined by the overall fold of the NusG NTD and is only weakly dependent on the nature of the individual amino acid side-chains, consistent with the mutational analysis of E. coli NusG (Mooney et al, 2009b).…”

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes

“…4C). Crickard et al recently reported that replacing six residues of Spt5, a yeast homolog of NusG, located on the same face as the SWHL NusG motif with alanines abolished Spt5 cross-linking to the NT DNA and its anti-arrest activity (31). Although it is uncertain if the altered surface residues (which are only weakly conserved) or more extensive structural changes (such as local refolding caused by altered core residues) lead to the observed phenotypes, it seems probable that the four-alanine substitution in NusG abolishes its anti-pausing activity by altering interactions with the NT DNA and not with the GL.…”

Structural visualization of the p53/RNA polymerase II assembly

“…In support of this idea, the elongation factor Spt4/5 targets the same position on the Pol II cleft as p53, displaying a closed conformation within the context of an elongation complex (Gnatt et al 2001;Kostek et al 2006;Bernecky et al 2016). Importantly, Spt4/5 directly interacts with the clamp domain to both positively and negatively regulate Pol II elongation (Yamaguchi et al 1999;Bernecky et al 2016;Crickard et al 2016).…”

“…This indicates that p53 could retain its ability to associate with DNA directly while in complex with Pol II. Perhaps this ability could allow p53 to mimic elongation factors Spt4/5 and NusG, which stabilize the upstream DNA and the nontemplate strand in the transcription bubble to help prevent Pol II backtracking (Herbert et al 2010;Chakraborty et al 2012;Bernecky et al 2016;Crickard et al 2016). Therefore, a conserved structural mechanism may be used by p53, Spt4/5, and NusG to modulate Pol II elongation activity.…”