Demeshkina N, Repkova M, Ven'yaminova A, Graifer D, Karpova G
Laboratory of Ribosomal Structure and Function and Group of Oligoribonucleotide Chemistry, Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of Russian Academy of Sciences.
RNA. 2000 Dec;6(12):1727-36. doi: 10.1017/s1355838200000996.
The 18S rRNA environment of the mRNA at the decoding site of human 80S ribosomes has been studied by cross-linking 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.
通过与六核苷酸UUUGUU（包含苯丙氨酸和缬氨酸密码子）的衍生物进行交联，研究了人80S核糖体解码位点处mRNA的18S rRNA环境。这些衍生物在鸟嘌呤的N7原子或5'-末端尿嘧啶残基的C5原子上带有全氟苯基叠氮基团。通过同源tRNA调整密码子在核糖体A、P或E位点的位置。对于每种类型的衍生物，均获得了三种类型的复合物，即：（1）P位点的密码子UUU和苯丙氨酸-tRNA苯丙氨酸（A位点的密码子GUU）；（2）P位点的密码子UUU和苯丙氨酸-tRNA苯丙氨酸以及A位点的苯丙氨酸缬氨酸-tRNA缬氨酸；（3）P位点的密码子GUU和缬氨酸-tRNA缬氨酸（E位点的密码子UUU）。这使得mRNA类似物的修饰核苷酸能够定位在核糖体上的-3、+1或+4位置。温和的紫外线照射导致mRNA类似物与18S rRNA发生tRNA依赖性交联。核苷酸G961与mRNA的-3位置交联，核苷酸G1207与+1位置交联，A1823与A1824一起与+4位置交联。所有这些核苷酸都位于小亚基RNA结构中最保守的区域，并且对应于细菌16S rRNA的核苷酸G693、G926、G1491和A1492。其中三个（G1491除外）先前已在70S核糖体解码位点被发现。讨论了16S和18S rRNA解码位点之间的异同。
