RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms a hairpin in the RfaH:ops complex structure, flipping out a conserved T residue that is specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the structure of the non-template strand are read out by transcription factors, expanding the repertoire of transcriptional regulators in all domains of life.

In the present study, two antioxidant micromolecular components (L2f-2 and L2f-3) and an antioxidant macromolecular component LB2 were extracted from lotus (Nelumbo nucifera Gaertn.) rhizomes. MS, FTIR (Fourier-transform IR) spectroscopy and NMR were used to identify these compounds. L2f-2 was (+/-)-gallocatechin, L2f-3 was (-)-catechin and LB2 was a polysaccharide-protein complex with a molecular mass of 18.8 kDa. LB2 was identified as a polysaccharide sulfate containing α/β-pyranose and α-furanose according to its FTIR spectrogram. It was composed of mannose, rhamnose, glucose, galactose and xylose with a molar ratio 2:8:7:8:1. The antioxidant components L2f-2, L2f-3 and LB2 strongly inhibited HIV-1 RT (reverse transcriptase) and IN (integrase). LB2 inhibited RT with an IC50 value of 33.7 μM. It also exhibited the highest HIV-1 3'-processing inhibitory activity with an IC50 value of 5.28 μM. Both L2f-2 and L2f-3 up-regulated the expression of IL-2 (interleukin-2) and down-regulated IL-10, while LB2 exhibited positive regulation on IL-2, IL-4 and IL-10. Moreover, L2f-3 and LB2 might inhibit HIV-1 directly by down-regulating TNFα (tumour necrosis factor α). These natural antioxidant components with antiviral and immunoregulatory activities could be potentially important for anti HIV-1 drug development and application to HIV-1 therapy.

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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...

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