Studying functional significance of the sequence 980-1061 in the central domain of human 18S rRNA using complementary DNA probes.
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(imet) 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(imet).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.
Graifer
D M
DM
Laboratory of Ribosomal Structure and Function, Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Russia.
Malygin
A A
AA
Matasova
N B
NB
Mundus
D A
DA
Zenkova
M A
MA
Karpova
G G
GG
D000478:Alkylation; D001483:Base Sequence; D001665:Binding Sites; D002474:Cell-Free System; D018076:DNA, Complementary; D006801:Humans; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D010452:Peptide Biosynthesis; D010455:Peptides; D011072:Poly U; D014176:Protein Biosynthesis; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000235:genetics*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D016914:Ribonuclease H; D012270:Ribosomes / Q000378:metabolism
D018076:DNA, Complementary; D009692:Nucleic Acid Heteroduplexes; D010455:Peptides; D012337:RNA, Ribosomal, 18S; D012346:RNA, Transfer, Amino Acyl; D011072:Poly U; C012001:polyphenylalanine; D016914:Ribonuclease H