Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region; a site-directed cross-linking study with mRNA analogues.
Synthetic mRNA analogues were prepared by T7 transcription, each containing several thio-uridine residues at selected positions. After binding to the ribosome in the presence of cognate tRNA, the thio-U residues were activated by UV irradiation and the resulting sites of cross-linking to 16S RNA analysed. Three distinct cross-links were consistently observed: (i) from position '+6' of the mRNA (the 3'-base of the A-site codon) to base 1052 of 16S RNA; (ii) from position '+7' of the mRNA to base 1395; and (iii) from '+11' to base 532. Individual yields of the cross-links were strongly dependent on the particular mRNA sequence in each case. The '+11/532' and '+6/1052' cross-links were always entirely tRNA-dependent, whereas the '+7/1395' cross-link was observed at lower intensity in the absence of tRNA. In the presence of a second (A-site bound) tRNA the +6/1052 cross-link was markedly reduced. A cross-link to the 1050 region was again observed when a message carrying a thio-U at position '+9' was translocated on the ribosome so as to bring the thio-U to position +6. Taken together, the data are incompatible with some current models both for the three-dimensional arrangement of 16S RNA and for the orientation of the tRNA-mRNA complex in the ribosome.
Dontsova
O
O
Department of Chemistry, Moscow State University, Russia.
Dokudovskaya
S
S
Kopylov
A
A
Bogdanov
A
A
Rinke-Appel
J
J
Jünke
N
N
Brimacombe
R
R
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D012333:RNA, Messenger / Q000138:chemical synthesis / Q000378:metabolism* / Q000528:radiation effects; D012336:RNA, Ribosomal, 16S / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D016914:Ribonuclease H; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D013891:Thiouridine; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D009843:Oligoribonucleotides; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; C037442:ribosomal protein S7; D013891:Thiouridine; D012343:RNA, Transfer; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H