The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand

Abstract

21RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique 22 mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function 23 depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template 24 DNA strand of the transcription bubble. We used structural and functional analyses to elucidate 25 the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate 26 RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms 27 a hairpin in the RfaH:ops complex structure, flipping out a conserved T residue that is 28 specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion 29 that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the 30 structure of the non-template strand are read out by transcription factors, expanding the 31 repertoire of transcriptional regulators in all domains of life. 32 33 72 (Kohler et al., 2017; Saxena et al., 2018). Following TEC dissociation, RfaH has been proposed 73 to regain the autoinhibited state (Tomar et al., 2013), thus completing the cycle. 74 A model of E. coli RfaH bound to Thermus thermophilus TEC was constructed by arbitrarily 75 threading the NT DNA (absent in the X-ray structure) through the TEC (Belogurov et al., 2007). 76 While subsequent functional analysis of RfaH supports this model (Belogurov et al., 2010), the 77 path of the NT DNA and the details of ops:RfaH interactions remain unclear. The NT DNA is 78 flexible in the TEC (Kang et al., 2017) and could be trapped in a state incompatible with 79 5 productive elongation; RfaH/NusG and yeast Spt5 have been proposed to constrain the NT 80 strand to increase processivity (Crickard et al., 2016; NandyMazumdar et al., 2016). Direct 81 contacts to the NT DNA have been demonstrated recently for Bacillus subtilis NusG (Yakhnin et 82 al., 2016) and Saccharomyces cerevisiae Spt5 (Crickard et al., 2016). 83 Here we combined structural and functional analyses to dissect RfaH:ops interactions. Our data 84 argue that ops plays two roles in RfaH recruitment: it halts RNAP to aid loading of RfaH and 85 makes specific contacts with RfaH-NTD. Strikingly, we found that a small hairpin extruded from 86 the NT DNA is required for RfaH recruitment, demonstrating how NT DNA flexibility could be 87 harnessed for transcriptional regulation in this and potentially many other systems. 88 127 pausing (Figure 2D and Figure 2 -figure supplement 1), consistent with their variability in the 128 consensus pause sequence (Figure 2A). Conversely, the G12C substitution eliminated the pause 129 at U11, making measurements of RfaH antipausing activity unreliable, but did not abrogate RfaH 130 recruitment (Figures 2C,D), suggesting that pausing at U11 is dispensable for RfaH binding 131 when RNAP is transcribing slowly. 132 Observations that RfaH is recruited to RNAP transcribing the G12C template raised a possibi...

Functional and Regulatory Roles of Fold-Switching Proteins

“…Cellular assays showed that RfaH:RNAP binding is weak, however, and requires supplementation of cellular lysate to activate RfaH (Artsimovitch and Landick, 2002). Furthermore, autoinhibited RfaH binds ops DNA (Zuber et al, 2018). Together, these results show that neither RNAP nor ops DNA alone can activate RfaH in vitro.…”

“…RfaH functions only in the presence of a short DNA consensus sequence called ops (operon polarity suppressor) (Artsimovitch and Landick, 2002). The non-template DNA ops sequence forms a hairpin that RfaH binds specifically (Zuber et al, 2018). This ops-site specificity distinguishes RfaH from NusG, which colocalizes with RNAP at most genes (Mooney et al, 2009), not just those containing ops.…”

Processive Antitermination

“…RfaH is specifically recruited to transcription elongation complexes by binding to the non-template DNA strand of the ops-element; this occurs during the lifetime of a programmed transcriptional pause (22). The ops-element forms both a consensus pause sequence as well as a DNA hairpin loop that makes specific, direct contacts to the RfaH NTD (79). RfaH and NusG are mutually exclusive, as both homologs share the same binding site on RNAP (80,81).…”

The C-terminal domain of transcription factor RfaH: Folding, fold switching and energy landscape

“…This entirely different folded state is stabilized by favorable interactions with the RfaH N-terminal domain (NTD) [23]. The switch in structure from all-α to all-β is triggered when RfaH binds to RNA polymerase in a paused state, which underpins RfaH's regulatory function in transcription and translation [24,35].…”