Protein L18 binds primarily at the junctions of helix II and internal loops A and B in Escherichia coli 5 S RNA. Implications for 5 S RNA structure.
Ribonuclease and chemical probes were used to investigate the binding sites of ribosomal protein L18 on Escherichia coli 5 S RNA using both end-labelling and reverse transcriptase procedures. The results, together with earlier data, were superimposed on a cylindrical projection of RNA double helices and most of the protection effects were found to cluster in the major groove at two sites located on one side of the RNA at the junctions of helix II with the adjoining internal loops A and B. The loop A/helix II junction was investigated using 5 S RNA mutants, produced by site-directed mutagenesis, that exhibited altered binding properties to L18. These results, together with those from a circular dichroism study of L18 complexed with the wild-type and different mutant RNAs, enabled us to assign an L18-induced conformational change to loop A. We infer that this change contributes to the co-operative binding of L5 to helix I, which may be reinforced by the binding of the very basic N-terminal peptide of L18 within the minor groove of helix I. A psoralen derivative formed a mono-addition product with U25 within loop B in the free RNA but not in the L18 complex. Moreover, the modified molecules were selected against in L18 binding experiments. Protection effects that occurred within the adjoining helix III and loop C were compatible with a tertiary interaction between loop C and loop B/helix III that could be stabilized by the L18 binding to the junction of helix II and loop B. Further support for a bipartite binding site derived from the finding that ethidium bromide molecules that are displaced from E. coli 5 S RNA by L18 intercalate both at the loop A/helix II junction and in loop B at the binding site of the psoralen derivative.
Egebjerg
J
J
Kemisk Institut, Aarhus Universitet, Denmark.
Christiansen
J
J
Brown
R S
RS
Larsen
N
N
Garrett
R A
RA
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli; D005453:Fluorescence; D011564:Furocoumarins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D011564:Furocoumarins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012341:RNA, Ribosomal, 5S; D012269:Ribosomal Proteins; C029609:ribosomal protein L18