Structure of 4.5S RNA in the signal recognition particle of Escherichia coli as studied by enzymatic and chemical probing.
The structure of 4.5S RNA, the Escherichia coli homologue of the signal recognition particle (SRP) RNA, alone and in the SRP complex with protein P48 (Ffh) was probed both enzymatically and chemically. The molecule is largely resistant against single strand-specific nucleases, indicating a highly base paired structure. Reactivity appears mainly in the apical tetraloop and in one of the conserved internal loops. Although some residues are found reactive toward dimethylsulphate and kethoxal in regions predicted to be unpaired by the phylogenetic secondary structure model of 4.5S RNA, generally the reactivity is low, and some residues in internal loops are not reactive at all. RNase V1 cleaves the RNA at multiple sites that coincide with predicted helices, although the cleavages show a pronounced asymmetry. The binding of protein P48 to 4.5S RNA results in a protection of residues in the apical part of the molecule homologous to eukaryotic SRP RNA (domain IV), whereas the cleavages in the conserved apical tetraloop are not protected. Hydroxyl radical treatment reveals an asymmetric pattern of backbone reactivity; in particular, the region encompassing nucleotides 60-82, i.e., the 3' part of the conserved domain IV, is protected. The data suggest that a bend in the domain IV region, most likely at the central asymmetric internal loop, is an important element of the tertiary structure of 4.5S RNA. Hyperchromicity and lead cleavage data are consistent with the model as they reveal the unfolding of a higher-order structure between 30 and 40 degrees C. Protection by protein P48 occurs in this region of the RNA and, more strongly, in the 5' part of domain IV (nt 26-50, most strongly from 35 to 49). It is likely that P48 binds to the outside of the bent form of 4.5S RNA.
Lentzen
G
G
Institut für Molekularbiologie, Universität Witten/Herdecke, Germany glentzen@umi-wh.de.
Moine
H
H
Ehresmann
C
C
Ehresmann
B
B
Wintermeyer
W
W
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D017665:Hydroxyl Radical; D007854:Lead; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D012329:RNA, Bacterial; D018271:Signal Recognition Particle; D007854:Lead; D017665:Hydroxyl Radical; D012260:Ribonucleases