FIGURE 4.

YchF preferentially binds to the 30 S ribosomal subunit. (A) The pooled 30 S, 50 S, and 70 S ribosomal fractions from wild-type cells or wild-type cells expressing a plasmid-borne copy of ychF (pBadYchF, induced with 0.002% arabinose) as shown in Figure 3, were analyzed by immune-detection with α-YchF antibodies. P correspond to the polysomal fractions and * indicates an unspecific band that is detected by the α-YchF antibody. Representative blots of several independent experiments are shown. (B) Crystal structure of Haemophilus influenzae YchF (pdb: 1JAL). The positions where para-benzoyl-L-phenylalanine (pBpa) was incorporated for in vivo site-directed cross-linking using the E. coli numbering, are indicated in red. (C–E) Cells expressing either wild-type (wt) YchF or YchF with pBpa incorporated at the indicated position were induced with 0.002% arabinose and UV exposed when indicated or kept in the dark. Cells were lysed and fractionated, and YchF and its potential cross-link partners were enriched by a single metal-affinity chromatography step. After SDS-PAGE and western blotting, the membrane was analyzed with α-YchF antibodies. Representative blots of at least three independent experiments are shown.