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Nucleotides of 18S rRNA surrounding mRNA codons at the human ribosomal A, P, and E sites: A crosslinking study with mRNA analogs carrying an aryl azide group at either the uracil or the guanine residue

Published online by Cambridge University Press:  27 December 2000

NATALIA DEMESHKINA ,
MARINA REPKOVA ,
ALIYA VEN'YAMINOVA ,
DMITRI GRAIFER  and
GALINA KARPOVA
NATALIA DEMESHKINA
Affiliation:
Laboratory of Ribosomal Structure and Function and Group of Oligoribonucleotide Chemistry, Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of Russian Academy of Sciences, 630090, Novosibirsk, Russia
MARINA REPKOVA
Affiliation:
Laboratory of Ribosomal Structure and Function and Group of Oligoribonucleotide Chemistry, Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of Russian Academy of Sciences, 630090, Novosibirsk, Russia
ALIYA VEN'YAMINOVA
Affiliation:
Laboratory of Ribosomal Structure and Function and Group of Oligoribonucleotide Chemistry, Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of Russian Academy of Sciences, 630090, Novosibirsk, Russia
DMITRI GRAIFER
Affiliation:
Laboratory of Ribosomal Structure and Function and Group of Oligoribonucleotide Chemistry, Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of Russian Academy of Sciences, 630090, Novosibirsk, Russia
GALINA KARPOVA
Affiliation:
Laboratory of Ribosomal Structure and Function and Group of Oligoribonucleotide Chemistry, Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of Russian Academy of Sciences, 630090, Novosibirsk, Russia
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Abstract

The 18S rRNA environment of the mRNA at the decoding site of human 80S ribosomes has been studied by crosslinking with derivatives of hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group either at the N7 atom of the guanine or at the C5 atom of the 5′-terminal uracil residue. The location of the codons on the ribosome at A, P, or E sites has been adjusted by the cognate tRNAs. Three types of complexes have been obtained for each type derivative, namely, (1) codon UUU and Phe-tRNAPhe at the P site (codon GUU at the A site), (2) codon UUU and tRNAPhe at the P site and PheVal-tRNAVal at the A site, and (3) codon GUU and Val-tRNAVal at the P site (codon UUU at the E site). This allowed the placement of modified nucleotides of the mRNA analog at positions −3, +1, or +4 on the ribosome. Mild UV irradiation resulted in tRNA-dependent crosslinking of the mRNA analogs to the 18S rRNA. Nucleotide G961 crosslinked to mRNA position −3, nucleotide G1207 to position +1, and A1823 together with A1824 to position +4. All of these nucleotides are located in the most strongly conserved regions of the small subunit RNA structure, and correspond to nucleotides G693, G926, G1491, and A1492 of bacterial 16S rRNA. Three of them (with the exception of G1491) had been found earlier at the 70S ribosomal decoding site. The similarities and differences between the 16S and 18S rRNA decoding sites are discussed.

Keywords

18S ribosomal RNAdecoding sitehuman ribosomesmRNA analogsphotocrosslinking
Type
Research Article
Information
RNA , Volume 6 , Issue 12 , December 2000 , pp. 1727 - 1736
Copyright
© 2000 RNA Society

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