Grant C M, Hinnebusch A G
Section on Molecular Genetics of Lower Eukaryotes, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Mol Cell Biol. 1994 Jan;14(1):606-18. doi: 10.1128/mcb.14.1.606-618.1994.
Translational control of the GCN4 gene involves two short open reading frames in the mRNA leader (uORF1 and uORF4) that differ greatly in the ability to allow reinitiation at GCN4 following their own translation. The low efficiency of reinitiation characteristic of uORF4 can be reconstituted in a hybrid element in which the last codon of uORF1 and 10 nucleotides 3' to its stop codon (the termination region) are substituted with the corresponding nucleotides from uORF4. To define the features of these 13 nucleotides that determine their effects on reinitiation, we separately randomized the sequence of the third codon and termination region of the uORF1-uORF4 hybrid and selected mutant alleles with the high-level reinitiation that is characteristic of uORF1. The results indicate that many different A+U-rich triplets present at the third codon of uORF1 can overcome the inhibitory effect of the termination region derived from uORF4 on the efficiency of reinitiation at GCN4. Efficient reinitiation is not associated with codons specifying a particular amino acid or isoacceptor tRNA. Similarly, we found that a diverse collection of A+U-rich sequences present in the termination region of uORF1 could restore efficient reinitiation at GCN4 in the presence of the third codon derived from uORF4. To explain these results, we propose that reinitiation can be impaired by stable base pairing between nucleotides flanking the uORF1 stop codon and either the tRNA which pairs with the third codon, the rRNA, or sequences located elsewhere in GCN4 mRNA. We suggest that these interactions delay the resumption of scanning following peptide chain termination at the uORF and thereby lead to ribosome dissociation from the mRNA.
GCN4基因的翻译调控涉及mRNA前导序列中的两个短开放阅读框（上游开放阅读框1和上游开放阅读框4），它们在自身翻译后允许在GCN4处重新起始翻译的能力上存在很大差异。上游开放阅读框4重新起始效率低的特性可以在一个杂交元件中重现，在该杂交元件中，上游开放阅读框1的最后一个密码子及其终止密码子下游10个核苷酸（终止区域）被上游开放阅读框4的相应核苷酸取代。为了确定这13个核苷酸中决定其对重新起始影响的特征，我们分别对上游开放阅读框1 - 上游开放阅读框4杂交体的第三个密码子和终止区域的序列进行随机化处理，并选择具有上游开放阅读框1特征的高水平重新起始的突变等位基因。结果表明，存在于上游开放阅读框1第三个密码子处的许多不同的富含A + U的三联体可以克服源自上游开放阅读框4的终止区域对GCN4处重新起始效率的抑制作用。高效重新起始与指定特定氨基酸或同工受体tRNA的密码子无关。同样，我们发现，在上游开放阅读框4衍生的第三个密码子存在的情况下，存在于上游开放阅读框1终止区域的各种富含A + U的序列可以恢复GCN4处的高效重新起始。为了解释这些结果，我们提出，上游开放阅读框1终止密码子侧翼的核苷酸与与第三个密码子配对的tRNA、rRNA或GCN4 mRNA其他位置的序列之间的稳定碱基配对可能会损害重新起始。我们认为，这些相互作用会延迟上游开放阅读框处肽链终止后扫描的恢复，从而导致核糖体从mRNA上解离。
