Eckert V, Kyriatsoulis A, Gassen H G
Institut für Biochemie, Technische Hochschule Darmstadt, Federal Republic of Germany.
Eur J Biochem. 1989 Jul 1;182(3):557-62. doi: 10.1111/j.1432-1033.1989.tb14863.x.
Although much attention was focused on the role of the 16S RNA in mRNA selection by the 30S ribosomal subunit no true consensus site has emerged as yet. Oligonucleotides such as GAGG, UGAU and CCAA which are complementary to the 3' end of the 16S RNA stimulate the AUG-dependent binding of fMet-tRNA to 30S subunits. If those tetranucleotides are used in combination or if the octanucleotides GAGGUGAU and UGAUCCAA are applied, the degree of stimulation remains unchanged. Effects are strictly dependent on the presence of initiation factor 2 (IF-2) and cannot be produced by using A4 or U4. With sequences covalently linked to the AUG as in CCAAAUG and UGAUCCAAAUG, the efficiency of the initiation complex formation decreases significantly as compared to AUG with UGAUCCAAAUG being the least efficient mRNA analogue. The pentadecanucleotide GAGGUGAUCCAAAUG, however, shows the highest efficiency in directing the binding of the fMet-tRNA to 30S subunits and is clearly superior to AUG. Initiation factor 2 (IF-2), which stimulates tRNA binding significantly with AUG and CCAAAUG, both in terms of slope and plateau values of the binding curves, does not effect the initial rate of tRNA binding to GAGGUGAUCCAAAUG. In another set of experiments, where GAGG and AUG are separated by oligo(U) or oligo(A) sequences, the effect of chain length was investigated. mRNA analogues with a spacer of 6-9 nucleotides show the highest binding efficiencies, with a U spacer being superior to an A spacer, indicating that a more flexible spacer favours tRNA binding.
尽管很多注意力都集中在16S RNA在30S核糖体亚基选择mRNA中的作用上，但尚未出现真正的共有序列。与16S RNA 3'端互补的寡核苷酸，如GAGG、UGAU和CCAA，能刺激fMet - tRNA与30S亚基的AUG依赖性结合。如果将这些四核苷酸组合使用，或者应用八核苷酸GAGGUGAU和UGAUCCAA，刺激程度保持不变。这些效应严格依赖于起始因子2（IF - 2）的存在，使用A4或U4则无法产生这些效应。对于像CCAAAUG和UGAUCCAAAUG这样与AUG共价连接的序列，与AUG相比，起始复合物形成的效率显著降低，其中UGAUCCAAAUG是效率最低的mRNA类似物。然而，十五核苷酸GAGGUGAUCCAAAUG在指导fMet - tRNA与30S亚基结合方面显示出最高效率，明显优于AUG。起始因子2（IF - 2）在结合曲线的斜率和平台值方面都能显著刺激tRNA与AUG和CCAAAUG的结合，但不影响tRNA与GAGGUGAUCCAAAUG结合的初始速率。在另一组实验中，GAGG和AUG被寡聚（U）或寡聚（A）序列隔开，研究了链长的影响。间隔6 - 9个核苷酸的mRNA类似物显示出最高的结合效率，U间隔优于A间隔，这表明更灵活的间隔有利于tRNA结合。
