Figure 5. RfaH-CTD is required for effects on translation and may interact with S10.

(A, B) Reporter assays using the translation-competent (A, pIA955) and defective (B, pIA1087) ops-lux operon constructs, which differ in sequence 5 nt upstream from the ATG codon, GAGGA and CACAC, respectively. The assays were performed in the rfaH deletion strain transformed with plasmids encoding RfaH variants under control of the P_BAD promoter, as before (Belogurov et al., 2010). The results are expressed as luminescence corrected for the cell densities of individual cultures.

(C) Expression of rfb operon (top) evaluated by qRT-PCR. Total RNA was isolated from ΔrfaH cells expressing WT or an altered RfaH variant and the absolute amount of wbbI mRNA was measured. In (A-C), errors (± standard deviation) were calculated from three independent experiments.

(D–G) ChIP-chip analysis of the protein-coding rfb and atp operons and non-coding rrnE, rnpB and ssrA genes, performed as described previously (Belogurov et al., 2009; Mooney et al., 2009a) with probe sets for the rfb (D), atp (E), rrnE (F), ssrA (G), and rnpB (G), transcription units (TUs). Cy3 signal (IP) from the DNA immunoprecipitated with monoclonal antibodies to RNAP (ß-subunit), NusA, or HA-epitope tag on NusG or polyclonal antibodies to RfaH, NusE (S10), or NusB was divided by Cy5 signal (input) from unenriched DNA collected prior to immunoprecipitation. The data for each target were quantile normalized against each other so that relative signal ratios could be compared, and plotted on a log₂ scale. The ratios of average NusE/RNAP and NusG/RNAP signals were 1.1 and 0.47 (rfb), 1.1 and 0,88 (atp), 0.98 and 1.1 (rrnE), 0.35 and 0.45 (rnpB), and 0.36 and 0.55 (ssrA). See also Figure S5.