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首页> 外文期刊>Journal of Molecular Biology >A Group II Intron-encoded Maturase Functions Preferentially In Cis and Requires Both the Reverse Transcriptase and X Domains to Promote RNA Splicing.
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A Group II Intron-encoded Maturase Functions Preferentially In Cis and Requires Both the Reverse Transcriptase and X Domains to Promote RNA Splicing.

机译：组II内含子编码的成熟酶功能最好在顺式中，并且需要逆转录酶和X结构域才能促进RNA剪接。

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Mobile group II introns encode proteins with both reverse transcriptase activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active structure of the intron RNA. Previous studies with the Lactococcus lactis Ll.LtrB intron suggested a model in which the intron-encoded protein binds first to a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of its own coding region, and then makes additional contacts with conserved catalytic core regions to stabilize the active RNA structure. Here, we developed an Escherichia coli genetic assay that links the splicing of the Ll.LtrB intron to the expression of green fluorescent protein and used it to study the in vivo splicing of wild-type and mutant introns and to delineate regions of the maturase required for splicing. Our results show that the maturase functions most efficiently when expressed in cis from the same transcript as the intron RNA. In agreement with previous in vitro assays, we find that the high-affinity binding site in DIVa is required for efficient splicing of the Ll.LtrB intron in vivo, but in the absence of DIVa, 6-10% residual splicing occurs by the direct binding of the maturase to the catalytic core. Critical regions of the maturase were identified by statistically analyzing ratios of missense to silent mutations in functional LtrA variants isolated from a library generated by mutagenic PCR ("unigenic evolution"). This analysis shows that both the reverse transcriptase domain and domain X, which likely corresponds to the reverse transcriptase thumb, are required for RNA splicing, while the C-terminal DNA-binding and DNA endonuclease domains are not required. Within the reverse transcriptase domain, the most critical regions for maturase activity include parts of the fingers and palm that function in template and primer binding in HIV-1 reverse transcriptase, but the integrity of the reverse transcriptase active site is not required. Biochemical analysis of LtrA mutants indicates that the N terminus of the reverse transcriptase domain is required for high-affinity binding of the intron RNA, possibly via direct interaction with DIVa, while parts of domain X interact with conserved regions of the catalytic core. Our results support the hypothesis that the intron-encoded protein adapted to function in splicing by using, at least in part, interactions used initially to recognize the intron RNA as a template for reverse transcription.
机译：流动的II组内含子编码的蛋白质既具有逆转录酶活性（在内含子迁移中起作用），又具有成熟酶活性（通过稳定内含子RNA的催化活性结构来促进RNA剪接）。先前对乳酸乳球菌Ll.LtrB内含子的研究提出了一个模型，其中内含子编码的蛋白首先与内含子亚结构域DIVa中的高亲和力结合位点结合，该结构在其自身编码区的开始处是特异结构，然后产生附加与保守的催化核心区域接触以稳定活性RNA结构。在这里，我们开发了一种将Ll.LtrB内含子的剪接与绿色荧光蛋白表达联系起来的大肠杆菌遗传测定，并用它来研究野生型和突变型内含子的体内剪接并描绘了所需成熟酶的区域用于拼接。我们的结果表明，当与内含子RNA相同的转录物中顺式表达时，成熟酶的功能最有效。与以前的体外测定一致，我们发现DIVa中的高亲和力结合位点是体内Ll.LtrB内含子有效剪接所必需的，但是在没有DIVa的情况下，直接剪接会产生6-10％的残留剪接成熟酶与催化核心的结合。通过统计分析从诱变PCR（“单基因进化”）产生的文库中分离的功能性LtrA变体中的错义突变与沉默突变的比率来鉴定成熟酶的关键区域。该分析表明，RNA剪接既需要逆转录酶结构域也可以使用逆转录酶结构域X（可能对应于逆转录酶拇指），而不需要C端DNA结合结构域和DNA核酸内切酶结构域。在逆转录酶结构域内，成熟酶活性最关键的区域包括手指和手掌的部分，这些部分在模板中起作用，并且在HIV-1逆转录酶中结合引物，但不需要逆转录酶活性位点的完整性。 LtrA突变体的生化分析表明，内含子RNA的高亲和力结合可能需要逆转录酶结构域的N端，这可能是通过与DIVa的直接相互作用而实现的，而结构域X的某些部分则与催化核心的保守区相互作用。我们的结果支持这样的假设，即内含子编码的蛋白质通过至少部分地使用最初用于识别内含子RNA的模板作为逆转录模板而适于在剪接中起作用。

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