Bulygin Konstantin, Baouz-Drahy Soria, Graifer Dmitri, Favre Alain, Karpova Galina
Institut Jacques Monod, Laboratoire de Photobiologie Moléculaire (CNRS-UMR 7033, BioMoCeTi) Universités Paris 6 et Paris 13, 75251 Paris Cedex 05, France.
Biochim Biophys Acta. 2009 Mar;1789(3):167-74. doi: 10.1016/j.bbagrm.2008.12.001. Epub 2008 Dec 11.
Long synthetic mRNAs were used to study the positioning of the E site codon, the 2nd and 3rd nucleotides of the A site bound codon and a nucleotide 3' of this codon with respect to the 18S rRNA in the human 80S ribosome. The mRNAs contained a GAC triplet coding for Asp and a single 4-thiouridine residue (s(4)U) upstream or downstream of the GAC codon. In the presence of tRNA(Asp), the GAC codon of the mRNAs was targeted to the ribosomal P site thus placing s(4)U in one of the following positions -3, -2, -1, +5, +6 or +7 with respect to the first nucleotide of the P site bound codon. It was found that mRNAs that bore s(4)U in positions +5 to +7 cross-linked to the 18S rRNA nucleotides C1696, C1698 and 1820-1825, the distribution of cross-links among these targets depending on the position of s(4)U. Cross-links of mRNAs containing s(4)U in positions -3 to -1 were found in the region 1699-1704 of the 18S rRNA. In the absence of tRNA, all mRNAs cross-linked only to C1696 and C1698. Absence of the cross-linked nucleotides C1696 and C1698 in the case of mRNAs containing s(4)U in positions -3 to -1 confirmed that tRNA(Asp) actually phased the mRNA on the ribosome.
长链合成mRNA被用于研究E位点密码子的定位，A位点结合密码子的第2和第3个核苷酸以及该密码子3'端的一个核苷酸相对于人80S核糖体中18S rRNA的位置。这些mRNA包含一个编码天冬氨酸的GAC三联体以及在GAC密码子上游或下游的单个4-硫尿苷残基（s(4)U）。在天冬氨酸tRNA存在的情况下，mRNA的GAC密码子被靶向核糖体P位点，从而使s(4)U相对于P位点结合密码子的第一个核苷酸处于以下位置之一：-3、-2、-1、+5、+6或+7。结果发现，在+5至+7位置带有s(4)U的mRNA与18S rRNA核苷酸C1696、C1698和1820 - 1825发生交联，这些靶点之间的交联分布取决于s(4)U的位置。在-3至-1位置含有s(4)U的mRNA的交联发生在18S rRNA的1699 - 1704区域。在没有tRNA的情况下，所有mRNA仅与C1696和C1698发生交联。在-3至-1位置含有s(4)U的mRNA的情况下，交联核苷酸C1696和C1698的缺失证实了天冬氨酸tRNA实际上使mRNA在核糖体上定相。
