Lin J H, Levin H L
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Cell Biol. 1998 Nov;18(11):6859-69. doi: 10.1128/MCB.18.11.6859.
An inverted repeat (IR) within the U5 region of the Rous sarcoma virus (RSV) mRNA forms a structure composed of a 7-bp stem and a 5-nucleotide (nt) loop. This U5-IR structure has been shown to be required for the initiation of reverse transcription. The mRNA of Tf1, long terminal repeat-containing retrotransposon from fission yeast (Schizosaccharomyces pombe) contains nucleotides with the potential to form a U5-IR stem-loop that is strikingly similar to that of RSV. The putative U5-IR stem-loop of Tf1 consists of a 7-bp stem and a 25-nt loop. Results from mutagenesis studies indicate that the U5-IR stem-loop in the mRNA of Tf1 does form and that it is required for Tf1 transposition. Although the loop is required for transposition, we were surprised that the specific sequence of the nucleotides within the loop was unimportant for function. Additional investigation indicates that the loss of transposition activity due to a reduction in the loop size to 6 nt could be rescued by increasing the GC content of the stem. This result indicates that the large loop in the Tf1 mRNA relative to that of the RSV allows the formation of the relatively weak U5-IR stem. The levels of Tf1 proteins expressed and the amounts of Tf1 RNA packaged into the virus-like particles were not affected by mutations in the U5-IR structure. However, all of the mutations in the U5-IR structure that caused defects in transposition produced low amounts of reverse transcripts. A unique feature in the initiation of Tf1 reverse transcription is that, instead of a tRNA, the first 11 nt of the Tf1 mRNA serve as the minus-strand primer. Analysis of the 5' end of Tf1 mRNA revealed that the mutations in the U5-IR stem-loop that resulted in defects in reverse transcription caused a reduction in the cleavage activity required to generate the Tf1 primer. Our results indicate that the U5-IR stems of Tf1 and RSV are conserved in size, position, and function.
劳氏肉瘤病毒（RSV）mRNA的U5区域内的反向重复序列（IR）形成了一个由7个碱基对的茎和一个5个核苷酸（nt）的环组成的结构。已证明该U5-IR结构是逆转录起始所必需的。来自裂殖酵母（Schizosaccharomyces pombe）的含长末端重复序列的逆转座子Tf1的mRNA含有一些核苷酸，它们有可能形成一个与RSV的U5-IR茎环惊人相似的结构。Tf1的推定U5-IR茎环由一个7个碱基对的茎和一个25个核苷酸的环组成。诱变研究结果表明，Tf1 mRNA中的U5-IR茎环确实形成，并且它是Tf1转座所必需的。尽管环对于转座是必需的，但我们惊讶地发现环内核苷酸的特定序列对功能并不重要。进一步的研究表明，通过增加茎的GC含量，可以挽救由于环大小减小到6个核苷酸而导致的转座活性丧失。这一结果表明，相对于RSV而言，Tf1 mRNA中的大环允许形成相对较弱的U5-IR茎。U5-IR结构中的突变并不影响所表达的Tf1蛋白水平以及包装到病毒样颗粒中的Tf1 RNA量。然而，所有导致转座缺陷的U5-IR结构突变都产生了少量的逆转录产物。Tf1逆转录起始的一个独特特征是，Tf1 mRNA的前11个核苷酸而非tRNA作为负链引物。对Tf1 mRNA 5'端的分析表明，导致逆转录缺陷的U5-IR茎环突变导致了产生Tf1引物所需的切割活性降低。我们的结果表明，Tf1和RSV的U5-IR茎在大小、位置和功能上是保守的。
