Molotkov M V, Graĭfer D M, Demeshkina N A, Repkova M N, Ven'iaminova A G, Karpova G G
Mol Biol (Mosk). 2005 Nov-Dec;39(6):999-1007.
Short mRNA analogues carrying a UUU triplet at the 5'-termini and a perfluorophenylazide group at either the N7 atom of the guanosine or the C5 atom of the uridine 3' of the triplet were applied to study positioning of mRNA 3' of the A site codon. Complexes of 80S ribosomes with the mRNA analogues were obtained in the presence of tRNAPhe that directed UUU codon to the P site and consequently provided placement of the nucleotide with cross-linker in positions +9 or +12 with respect to the first nucleotide of the P site bound codon. Both types mRNA analogues cross-linked to the 18S rRNA and 40S proteins under mild UV-irradiation. Cross-linking patterns in the complexes where modified nucleotides of the mRNA analogues were in position +7 were analyzed for comparison (cross-linking to the 18S rRNA in such complexes has been studied previously). The efficiency of cross-linking to the ribosomal components depended on the nature of the modified nucleotide in the mRNA analogue and its position on the ribosome, extent of cross-linking to the 18S rRNA being decreased drastically when the modified nucleotide was moved from position +7 to position +12. The nucleotides of 18S rRNA cross-linked to mRNA analogues were determined. Modified nucleotides in positions +9 and +12 cross-linked to the invariant dinucleotide A1824/A1825 and to variable A1823 in the 3'-minidomain of 18S rRNA as well as to protein S15. The same ribosomal components have been found earlier to cross-link to modified mRNA nucleotides in positions from +4 to +7. Besides, all mRNA analogues cross-linked to the invariant nucleotide c1698 in the 3'-minidomain and to and the conserved region 605-620 closing helix 18 in the 5'-domain.
携带位于5'末端的UUU三联体以及位于三联体中鸟苷的N7原子或尿苷3'的C5原子上的全氟苯基叠氮基团的短mRNA类似物，被用于研究A位点密码子下游mRNA的定位。在将UUU密码子导向P位点的苯丙氨酰tRNA存在的情况下，获得了80S核糖体与mRNA类似物的复合物，从而使得带有交联剂的核苷酸相对于P位点结合密码子的第一个核苷酸处于+9或+12位。在温和紫外线照射下，两种类型的mRNA类似物均与18S rRNA和40S蛋白质发生交联。为了进行比较，分析了mRNA类似物的修饰核苷酸处于+7位的复合物中的交联模式（此前已研究过此类复合物中与18S rRNA的交联）。与核糖体组分的交联效率取决于mRNA类似物中修饰核苷酸的性质及其在核糖体上的位置，当修饰核苷酸从+7位移至+12位时，与18S rRNA的交联程度急剧下降。确定了与mRNA类似物交联的18S rRNA的核苷酸。处于+9和+12位的修饰核苷酸与18S rRNA 3'-小结构域中的不变二核苷酸A1824/A1825以及可变的A1823以及蛋白质S15发生交联。此前已发现相同的核糖体组分与处于+4至+7位的修饰mRNA核苷酸发生交联。此外，所有mRNA类似物均与3'-小结构域中的不变核苷酸c1698以及5'-结构域中封闭螺旋18的保守区域605-620发生交联。
