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

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• 作者：Graifer ; Dmitri M. ; Malygin ; Alexey A. ; Matasova ; Natalie B. ; Mundus ; Dmitri A. ; Zenkova ; Marina A. ; et. al.
• 关键词：Human ribosome ; Ribosomal function ; cDNA probe ; 18S rRNA
• 刊名：Biochimica et Biophysica Acta (BBA)/Gene Structure and Expression
• 出版年：1997
• 出版时间：March 28, 1997
• 年：1997
• 卷：1350
• 期：3
• 页码：335-344
• 全文大小：721 K

文摘

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^Met_i 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^Met_i · 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.