Mutational analysis of the mRNA operator for T4 DNA polymerase.
Mutational analysis of the mRNA operator for T4 DNA polymerase.
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T4 DNA 聚合酶的 mRNA 操纵子的突变分析。
DOI：
10.1093/genetics/128.2.203
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发表时间：
1991
期刊：
影响因子：
3.3
通讯作者：
Karam,JD
中科院分区：
文献类型：
--
作者：
Andrake,MD;Karam,JD
噬菌体 T4 DNA 聚合酶的生物合成在翻译水平上受到自体调节。该酶是基因 43 的产物，通过在 36-40 个核苷酸片段上结合其 mRNA 5' 起始子 AUG 来抑制自身翻译，该片段包括 Shine-Dalgarno 序列和由 5 碱基对茎和 8 碱基环组成的推定 RNA 发夹结构。我们构建了破坏茎或改变发夹特定环残基的突变，并发现其中许多突变，包括环序列中的单碱基变化、体外纯化的 T4 DNA 聚合酶与其 RNA 的结合减少（通过凝胶阻滞测定测量）以及体内酶合成的去抑制（通过 T4 感染和重组质粒介导的表达测量）。然而，体外效应并不总是与体内效应一致。例如，茎与野生型以外的序列配对导致体外蛋白质结合正常，但体内蛋白质合成去抑制。类似地，环中的C----A变化在体外具有较小的影响，但在体内具有较强的影响。相比之下，发夹基部附近的 A----U 变化预计会增加碱基配对茎的长度，但在体外和体内效果都较小。结果表明，T4 DNA 聚合酶与其结构化 RNA 操纵子的相互作用取决于特定核苷酸残基的空间排列，并且在体内受到调节。
Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level. The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop. We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression). In vitro effects, however, were not always congruent with in vivo effects. For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo. Similarly, a C----A change in the loop had a small effect in vitro and a strong effect in vivo. In contrast, an A----U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo. The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo.