mRNA Binding Track in the Human 80S Ribosome for mRNA Analogs Randomly Substituted with 4-Thiouridine Residues

• Published in: Biochemistry (Easton) 1994-05, Vol.33 (20), p.6201-6206
• Main Authors: Graifer, Dmitri M, Juzumiene, Dalia I, Karpova, Galina G, Wollenzien, Paul
• Format: Article
• Language: English
• Subjects: Base Sequence; Binding Sites; Cross-Linking Reagents; Electrophoresis, Agar Gel; Humans; Molecular Sequence Data; Nucleic Acid Conformation; Ribosomes - metabolism; RNA, Messenger - chemistry; RNA, Messenger - metabolism; RNA, Ribosomal, 18S - metabolism; RNA, Transfer, Phe - metabolism; RNA, Transfer, Trp - metabolism; RNA-Directed DNA Polymerase - metabolism; Thiouridine; Ultraviolet Rays
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00186a020

The interaction between mRNA and 18S rRNA in human 80S ribosomes has been studied using synthetic mRNA analogues randomly substituted with 4-thiouridine, which can be photoactivated for cross-linking. Two mRNA analogues with different sequences have been used for complex formation with ribosomes without or with the presence of a cognate tRNA. Cross-linked 18S rRNA nucleotides were identified by reverse transcription analysis. The base U630 in 18S rRNA was the main target of cross-linking for both of the mRNA analogues studied, and three minor sites of cross-linking, A1060, U1046, and U966, were also identified. Thus, in the case of human 80S ribosomes, the set of nucleotide residues cross-linked to the mRNA analogues is significantly smaller than the twelve sites seen for Escherichia coli with these same two mRNA analogues [Bhangu, R., & Wollenzien, P. (1992) Biochemistry 31, 5937-5944]. The residue U630 is within a highly conserved region corresponding to the 530 loop region of eubacterial 16S rRNA; the cross-link to this site indicates that it plays a key role in interacting with mRNA on 80S ribosomes independently of the presence of a cognate tRNA at the P site.