Site-directed cross-linking of mRNA analogues to 16S ribosomal RNA; a complete scan of cross-links from all positions between '+1' and '+16' on the mRNA, downstream from the decoding site.
mRNA analogues containing 4-thiouridine residues at selected sites were used to extend our analysis of photo-induced cross-links between mRNA and 16S RNA to cover the entire downstream range between positions +1 and +16 on the mRNA (position +1 is the 5'-base of the P-site codon). No tRNA-dependent cross-links were observed from positions +1, +2, +3 or +5. Position +4 on the mRNA was cross-linked in a tRNA-dependent manner to 16S RNA at a site between nucleotides ca 1402-1415 (most probably to the modified residue 1402), and this was absolutely specific for the +4 position. Similarly, the previously observed cross-link to nucleotide 1052 was absolutely specific for the +6 position. The previously observed cross-links from +7 to nucleotide 1395 and from +11 to 532 were however seen to a lesser extent with certain types of mRNA sequence from neighbouring positions (+6 to +10, and +10 to +13, respectively); no tRNA-dependent cross-links to other sites on 16S RNA were found from these positions, and no cross-linking was seen from positions +14 to +16. In each case the effect of a second cognate tRNA (at the ribosomal A-site) on the level of cross-linking was studied, and the specificity of each cross-link was confirmed by translocation experiments with elongation factor G, using appropriate mRNA analogues.
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, Germany.
J
J
Rinke-Appel
N
N
Jünke
R
R
Brimacombe
S
S
Dukudovskaya
O
O
Dontsova
A
A
Bogdanov
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D008969:Molecular Sequence Data; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000494:pharmacology; D010777:Photochemistry; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000494:pharmacology; D012365:RNA, Transfer, Tyr / Q000494:pharmacology; D016914:Ribonuclease H / Q000378:metabolism; D013891:Thiouridine / Q000737:chemistry
D003432:Cross-Linking Reagents; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012358:RNA, Transfer, Met; D012365:RNA, Transfer, Tyr; D013891:Thiouridine; D016914:Ribonuclease H