Rinke-Appel J, Jünke N, Brimacombe R, Lavrik I, Dokudovskaya S, Dontsova O, Bogdanov A
Max-Planck-Institut für Molekulare Genetik, Berlin, Germany.
Nucleic Acids Res. 1994 Aug 11;22(15):3018-25. doi: 10.1093/nar/22.15.3018.
mRNA analogues containing several 4-thiouridine (thio-U) residues at selected positions were prepared by T7-transcription. The spacer region between the Shine-Dalgarno sequence and the AUG codon consisted of four or eight bases with a single thio-U at a variable position; alternatively, cro-mRNA analogues were used carrying the thio-U substituted spacer sequence UUGU. The mRNAs were bound to E. coli ribosomes, and--after irradiation--the sites of cross-linking to 16S RNA were analysed. Three cross-links to the 16S RNA from the spacer region were observed, namely to positions 665, 1360, and a site close to nucleotide 1530. The cross-links were formed in different amounts in the presence or absence of tRNA(fMet), and were observed from thio-U residues located at various positions within the spacer sequence, although in the presence of tRNA they were in general stronger from positions close to the Shine-Dalgarno end of the spacer. The cross-linking behaviour in this upstream area of the mRNA is thus rather different in character from the previously published pattern in the downstream area. From considerations of structural conservation in small subunit RNA, we propose that both the upstream and downstream cross-links to 16S RNA reflect a universal mRNA path through the ribosome.
通过T7转录制备了在选定位置含有多个4 - 硫尿苷（硫代 - U）残基的mRNA类似物。Shine - Dalgarno序列与AUG密码子之间的间隔区由四个或八个碱基组成，在可变位置有一个硫代 - U；或者，使用携带硫代 - U取代的间隔序列UUGU的cro - mRNA类似物。将这些mRNA与大肠杆菌核糖体结合，照射后分析与16S RNA的交联位点。观察到间隔区与16S RNA有三个交联位点，分别位于665、1360位以及靠近核苷酸1530的一个位点。在有或没有起始tRNA（fMet）的情况下，交联的形成量不同，并且在间隔序列内不同位置的硫代 - U残基处都观察到了交联，尽管在有起始tRNA的情况下，通常从靠近间隔区Shine - Dalgarno末端的位置形成的交联更强。因此，mRNA这个上游区域的交联行为在性质上与先前发表的下游区域模式有很大不同。基于小亚基RNA结构保守性的考虑，我们提出与16S RNA的上游和下游交联都反映了mRNA穿过核糖体的通用路径。
