Demeshkina Natalia, Laletina Elena, Meschaninova Maria, Ven'yaminova Aliya, Graifer Dmitri, Karpova Galina
Laboratory of Ribosomal Structure and Function, and Group of Oligoribonucleotide Chemistry, Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva, 8, 630090, Novosibirsk, Russia.
Biochim Biophys Acta. 2003 May 13;1627(1):39-46. doi: 10.1016/s0167-4781(03)00072-1.
Positioning of each nucleotide of the E site and the P site bound codons with respect to the 18S rRNA on the human ribosome was studied by cross-linking with mRNA analogs, derivatives of the hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group on the second or the third uracil, and a derivative of the dodecaribonucleotide UUAGUAUUUAUU with a similar group on the guanine residue. The location of the modified nucleotides at any mRNA position from -3 to +3 (position +1 corresponds to the 5' nucleotide of the P site bound codon) was adjusted by the cognate tRNAs. A modified uridine at positions from -1 to +3 cross-linked to nucleotide G1207 of the 18S rRNA, and to nucleotide G961 when it was in position -2. A modified guanosine cross-linked to nucleotide G1207 if it was in position -3 of the mRNA. These data indicate that nucleotide G961 of the 18S rRNA is close only to mRNA positions -3 and -2, while G1207 is in the vicinity of positions from -3 to +3. The latter suggests that there is a sharp turn between the P and E site bound codons that brings nucleotide G1207 of the 18S rRNA close to each nucleotide of these codons. This correlates well with X-ray crystallographic data on bacterial ribosomes, indicating existence of a sharp turn between the P site and E site bound codons near a conserved nucleotide G926 of the 16S rRNA (corresponding to G1207 in 18S rRNA) close to helix 23b containing the conserved nucleotide 693 of the 16S rRNA (corresponding exactly to G961 of the 18S rRNA).
通过与mRNA类似物交联，研究了人核糖体上E位点和P位点结合密码子的每个核苷酸相对于18S rRNA的定位。这些mRNA类似物是六核糖核苷酸UUUGUU（包含苯丙氨酸和缬氨酸密码子）的衍生物，在第二个或第三个尿嘧啶上带有全氟苯基叠氮基团，以及十二核糖核苷酸UUAGUAUUUAUU的衍生物，在鸟嘌呤残基上带有类似基团。通过同源tRNA调节修饰核苷酸在从-3到+3的任何mRNA位置的定位（位置+1对应于P位点结合密码子的5'核苷酸）。在-1到+3位置的修饰尿苷与18S rRNA的核苷酸G1207交联，当它处于-2位置时与核苷酸G961交联。如果修饰鸟苷处于mRNA的-3位置，则与核苷酸G1207交联。这些数据表明，18S rRNA的核苷酸G961仅靠近mRNA位置-3和-2，而G1207在-3到+3位置附近。后者表明，在P位点和E位点结合的密码子之间存在急剧转弯，使18S rRNA的核苷酸G1207靠近这些密码子的每个核苷酸。这与细菌核糖体的X射线晶体学数据很好地相关，表明在靠近包含16S rRNA保守核苷酸693（与18S rRNA中的G961完全对应）的螺旋23b的16S rRNA保守核苷酸G926（与18S rRNA中的G1207对应）附近，P位点和E位点结合的密码子之间存在急剧转弯。
