Studying functional significance of the sequence 980–1061 in the central domain of human 18S rRNA using complementary DNA probes

Abstract

Region 980–1061 in human 18S rRNA has been chosen on the basis of our previous results, indicating that cross-linking sites of the alkylating mRNA analogs are located within this region. In the present study, we have used 10 DNA 15-mers complementary to various overlapping sequences within the 18S rRNA positions 980–1061. Their abilities to bind selectively to the target rRNA sequences were proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Four sequences (980–994, 987–1001, 1025–1039 and 1032–1046) were found to be well accessible for binding of the respective cDNA probes within 40S subunits. None of the oligomers inhibited tRNA Phe-dependent binding of oligo(U) messenger to 40S subunits and binding of Met-tRNA i Met to 40S subunits in the presence of eIF-2 and nonhydrolysable GTP analog. Nevertheless, two probes (complementary to the 18S rRNA sequences 987–1001 and 1025–1039 (being covalently attached to 40S subunits, inhibited translation of poly(U) by human 80S ribosomes in a cell-free system. The same oligomers revealed the most pronounced inhibitory action on the binding of messenger trinucleotide in the complex pAUG · 40S · Met-tRNA i Met· eIF-2 · GTP. Results of these functional assays demonstrate the importance of the 18S rRNA sequences 987–1001 and 1025–1039 for translation process on human ribosomes, most probably at the initiation step.