Structural analysis of the 5′ domain of the HeLa 18S ribosomal RNA by chemical and enzymatic probing

Abstract

The secondary structure of HeLa 18S rRNA was investigated by a combination of chemical and enzymatic probing techniques. Using four chemical reagents (DMS*, kethoxal, DEPC and CMCT) which react specifically with unpaired bases and two nucleases (RNase T1 and cobra venom nuclease) which cleave the ribopolynucleotides at unpaired guanines and helical segments, we have analyzed the secondary structure of the 5' domain of 18S rRNA isolated from HeLa 40S ribosomal subunits. The sites at which chemical modifications and nuclease cleavages occurred were identified by primer extension using synthetic deoxyoligonucleotides and reverse transcriptase. These studies led to the deduction of an intra-RNA pairing pattern from the available secondary structure models based on comparative sequence analysis. Apart from the general canonical pairing we have identified noncanonical U-U, G-A, A-G, A-C, C-A and G-G pairing in HeLa 18S rRNA. The differential reactivity of bases to chemical reagents has enabled us to predict the possible configuration of these bases in some of the noncanonical pairing. The absence of chemical reactivities and cobra venom nuclease sensitivity in the terminal loops of helices 6 and 12 indicate a tertiary interaction unique to HeLa 18S rRNA. We have confirmed the existence of the complex tertiary folding recently proposed (Gutell and Woese 1990 Proc. Natl. Acad. Sci. 87, 663-667) for the universally conserved helix 19 in HeLa 18S rRNA. The complementarity of chemical modifications and enzymatic cleavages provided experimental evidence for the proposal of a model structure for the 655 nucleotides of the 5' domain of HeLa 18S rRNA.