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Figure 4.
Figure 4. BoxA RNA Binding
(A) Mapping of crosslinked
peptides on the surface of the NusB-S10^Δloop complex. The view
is from the top of Figure 2A. NusB, dark gray; S10, light gray.
Crosslinked peptides of NusB (B1, B2, B3; see Table S3 for
peptide sequences) are dark blue, cyan, and steel blue,
respectively. Crosslinked peptides of S10 (E1 and E3) are red
and violet, respectively. Asp118, gold. RNAs encompassing the
rrn and λ BoxA elements and used for crosslinking are given
above and below the structure, respectively. Boxed regions with
residue numbers indicate the core BoxA elements. Residues in
green of rrn BoxA RNA and λ BoxA RNA have previously been
implicated in recruitment of NusB and S10 to antitermination
complexes by mutational analysis (Mogridge et al., 1998).
Outlined residues differ in λ BoxA compared to rrn BoxA. Black
bars designate crosslinked regions of the RNAs. They are
connected by lines to the peptides, to which they have been
crosslinked (Table S3). Inset 1 illustrates the deduced topology
of the NusB-S10-BoxA RNA complexes.
(B) (Top)
Representative crosslinking of λ NutR BoxA RNA (left two
panels) or rrn BoxA RNA (right two panels) to NusB-S10^Δloop or
NusB101-S10^Δloop (NusB^Asp118Asn-S10^Δloop). Two
concentrations of protein complex (0.31 and 0.62 μM) were
crosslinked, resolved on SDS gels, and visualized by
autoradiography. In each panel, RNA alone is in the left lane,
NusB-S10^Δloop complex in the central lane, and
NusB101-S10^Δloop complex in the right lane. (Bottom)
Quantification of crosslink yields. Values are the crosslink
yields of the protein components of the NusB101-S10^Δloop
samples, relative to the crosslink yields of the corresponding
components of the NusB-S10^Δloop samples. The crosslink yields
of the components of the NusB-S10^Δloop samples were set at
100% (dashed lines). Values represent the mean of three
independent experiments ± the standard errors of the
mean. ^*p ≤ 0.032; ^**p ≤ 0.020.
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