The environment of 5S rRNA in the ribosome: cross-links to the GTPase-associated area of 23S rRNA.
Two photoreactive diazirine derivatives of uridine were used to study contacts between 5S rRNA and 23 rRNA in situ in Escherichia coli ribosomes. 2'-Amino-2'-deoxy-uridine or 5-methyleneaminouridine were introduced into 5S rRNA by T7 transcription. After incorporation of these uridine analogues into the transcript their amino groups were modified with 4-[3-(trifluoromethyl)-3 H -diazirin-3-yl]benzyl isothiocyanate or the N -hydroxysuccinimide ester of 4-[3-(trifluoromethyl)-3 H -diazirin-3-yl]benzoic acid respectively. 5S rRNA carrying the photoreactive diazirine groups (referred to as the 2'-aminoribose derivative and the 5-methyleneamino derivative respectively) was reconstituted into 50S subunits or 70S ribosomes. After mild UV irradiation cross-links formed to 23S rRNA were analysed by standard procedures. All of the observed cross-links involved residue U89 of the 5S rRNA. Three nucleotides of 23S rRNA were cross-linked to this residue with the 5-methyleneamino derivative, namely U958, G1022 and G1138. With the 2'-aminoribose derivative a single cross-link was found, to U958. The significance of these cross-links for our understanding of the structure and function of 5S rRNA and its environment in the ribosome are discussed.
Sergiev
P
P
A. N. Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Moscow State University, Moscow 119899, Russia.
Dokudovskaya
S
S
Romanova
E
E
Topin
A
A
Bogdanov
A
A
Brimacombe
R
R
Dontsova
O
O
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003857:Deoxyuridine; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014529:Uridine / Q000031:analogs & derivatives; D014544:Uridine Triphosphate / Q000031:analogs & derivatives
C105886:5-methyleneaminouridine triphosphate; D003432:Cross-Linking Reagents; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D012341:RNA, Ribosomal, 5S; D020558:GTP Phosphohydrolases; D014544:Uridine Triphosphate; D003857:Deoxyuridine; D014529:Uridine