REQUIREMENTS FOR INTERCISTRONIC DISTANCE AND LEVEL OF EUKARYOTIC INITIATION FACTOR-II ACTIVITY IN REINITIATION ON GCN4 MESSENGER-RNA VARY WITH THE DOWNSTREAM CISTRON
REQUIREMENTS FOR INTERCISTRONIC DISTANCE AND LEVEL OF EUKARYOTIC INITIATION FACTOR-II ACTIVITY IN REINITIATION ON GCN4 MESSENGER-RNA VARY WITH THE DOWNSTREAM CISTRON
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DOI：
10.1128/mcb.14.4.2616
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发表时间：
1994-04-01
影响因子：
5.3
通讯作者：
HINNEBUSCH, AG
中科院分区：
文献类型：
--
作者：
GRANT, CM;MILLER, PF;HINNEBUSCH, AG
GCN 4基因的翻译控制是由GCN 4 mRNA前导序列中的四个短开放阅读框（uORF）和真核起始因子2（eIF-2）的磷酸化介导的。我们已经提出，通过磷酸化其α亚基或通过eIF-2再循环因子eIF-2B中的突变来降低eIF-2活性，使得已经翻译了5 '-近端uOPF 1的核糖体绕过uORF 2到uORF 4，并在GCN 4处重新起始。在这份报告中，我们提出了两条证据，证明所有合成GCN 4的核糖体先前都翻译了uORF 1，恢复了扫描，并在GCN 4起始位点重新启动。首先，当uORF 1被延长以使其与GCN 4编码区的起始部分重叠时，GCN 4表达被消除。第二，随着uORF 1逐渐靠近GCN 4，GCN 4的表达降低，当uORF 1与GCN 4之间只有32个核苷酸时，GCN 4的表达水平降低到所有uORF缺失时的5%。我们还发现，在uORF 4和GCN 4之间插入小的合成uORFs在去抑制条件下抑制了GCN 4的表达，证实了在eIF-2活性降低的条件下GCN 4的再起始与uORF 1下游再起始位点的距离成比例的想法。虽然uORF 4和GCN 4在捕获从mRNA的5'帽下游扫描的核糖体方面似乎同样有效，但这两个ORF在捕获从uORF 1扫描的再起始核糖体的能力方面差异很大。当eIF-2的活性形式以高水平存在时，在uORF 4处的再起始似乎比在GCN 4处更有效，当每个都非常接近uORF 1时。在eIF-2的循环减少的条件下，uORF 4的再起始基本上被抑制，这使得核糖体能够到达GCN 4起始位点;相反，缺乏uORF 4的构建体中GCN 4的再起始不受eIF-2活性水平降低的影响。这最后一个发现提出了这样的可能性，即除了eIF-2-GTP-Met-tRNA（i）Met三元复合物之外，第二因子与核糖体的时间依赖性结合是GCN 4处再起始的速率限制。此外，我们的研究结果表明，翻译再起始的效率可以强烈影响下游顺反子的性质以及顺反子间的距离。
Translational control of the GCN4 gene in response to amino acid availability is mediated by four short open reading frames in the GCN4 mRNA leader (uORFs) and by phosphorylation of eukaryotic initiation factor 2 (eIF-2). We have proposed that reducing eIF-2 activity by phosphorylation of its alpha subunit or by a mutation in the eIF-2 recycling factor eIF-2B allows ribosomes which have translated the 5'-proximal uOPF1 to bypass uORF2 to uORF4 and reinitiate at GCN4 instead. In this report, we present two lines of evidence that all ribosomes which synthesize GCN4 have previously translated uORF1, resumed scanning, and reinitiated at the GCN4 start site. First, GCN4 expression was abolished when uORF1 was elongated to make it overlap the beginning of the GCN4 coding region. Second, GCN4 expression was reduced as uORF1 was moved progressively closer to GCN4, decreasing to only 5% of the level seen in the absence of all uORFs when only 32 nucleotides separated uORF1 from GCN4. We additionally found that inserting small synthetic uORFs between uORF4 and GCN4 inhibited GCN4 expression under derepressing conditions, confirming the idea that reinitiation at GCN4 under conditions of diminished eIF-2 activity is proportional to the distance of the reinitiation site downstream from uORF1. While uORF4 and GCN4 appear to be equally effective at capturing ribosomes scanning downstream from the 5' cap of mRNA, these two ORFs differ greatly in their ability to capture reinitiating ribosomes scanning from uORF1. When the active form of eIF-2 is present at high levels, reinitiation appears to be much more efficient at uORF4 than at GCN4 when each is located very close to uORF1. Under conditions of reduced recycling of eIF-2, reinitiation at uORF4 is substantially suppressed, which allows ribosomes to reach the GCN4 start site; in contrast, reinitiation at GCN4 in constructs lacking uORF4 is unaffected by decreasing the level of eIF-2 activity. This last finding raises the possibility that time-dependent binding to ribosomes of a second factor besides the eIF-2-GTP-Met-tRNA(i)Met ternary complex is rate limiting for reinitiation at GCN4. Moreover, our results show that the efficiency of translational reinitiation can be strongly influenced by the nature of the downstream cistron as well as the intercistronic distance.