Chemical probing of the conformation of the 3'-functional domain of rabbit 18S rRNA in 40S subunits, 80S monosomes, and polysomes

Ribosomal RNA is becoming increasingly important in our current understanding of the translational process. The functional role of RNA has been shown to be closely correlated to its -molecular structure, whereby biological processes are directed by structural features within the RNA molecule. Recent evidence suggests that the 3'-minor domain of eukaryotic 18S rRNA, as in prokaryotes, is directly involved in protein biosynthesis. To begin to understand the functional organization of the eukaryotic 40S subunit, the primary and secondary structures of its naked 18S rRNA were determined. The nucleotide sequence of rabbit 18S rRNA was revised using dideoxy sequencing methodology. Chemical probing of adenosine residues using diethylpyrocarbonate and dimethyl sulfate, which specifically react with unpaired nucleotides, provided experimental evidence generally confirming the secondary structure model of 18S rRNA based on comparative sequence analysis. To reveal regions of possible functional importance in the 3'-minor domain, the higher order structure of 18S rRNA in 40S subunits, 80S monosomes and polyribosomes was compared by chemical footprinting with diethylpyrocarbonate and dimethyl sulfate. The modified 18S was isolated from these ribonucleoprotein particles and the resultant modified nucleotides identified on polyacrylamide sequencing gels upon either aniline-induced strand scission of 32P-end-labeled intact rRNA or by DNA-primer extension using sequence specific deoxyoligonucleotides with reverse transcriptase. Nucleotides C1696, G1697, A1817, A1818 and G1831 exhibited reduced reactivity toward chemical probes in translating ribosomes, implicating these residues as having functional significance. This finding is consistent with other studies suggesting that the regions 1690-1703 and 1818-1832 play active roles in the translational process