Photoaffinity labeling of the primer binding domain in murine leukemia virus reverse transcriptase.
Photoaffinity labeling of the primer binding domain in murine leukemia virus reverse transcriptase.
复制标题
鼠白血病病毒逆转录酶中引物结合域的光亲和标记。
DOI：
10.1021/bi00240a014
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Modak,MJ
中科院分区：
文献类型：
--
作者：
Tirumalai,RS;Modak,MJ
摘要：我们利用紫外光能量，将标记为d(T)8的5‘端共价连接到小鼠白血病病毒逆转录酶(MuLV RT)上，标记了MuLV RT的引物结合域。结果表明：(1)其他寡聚物、tRNAs和模板引物很容易与放射性标记的d(T)8竞争进行交联反应；(2)在相似条件下，竞争引物和模板引物对MuLV RT的DNA聚合酶活性也有相似程度的抑制作用；(3)底物脱氧核苷酸不起作用；(4)反应对高离子强度敏感。为了确定MuLV RT中的引物结合结构域/位点，将共价交联的MuLV RT和[32P]d(T)8复合物制备的胰酶消化产物在C-18柱上进行了反相高效液相色谱分离。三个不同的放射性标记的多肽被发现含有大部分的边界引物。这些多肽的氨基酸组成和序列分析表明，多肽I跨越MuLV RT一级氨基酸序列中的72-80个氨基酸残基，位于聚合酶区域。引物的交联点似乎位于Pro-76或其附近。多肽II和III分别跨越602-609和615-622个氨基酸残基，位于核糖核酸酶H区。IV小鼠白血病病毒逆转录酶(MuLV RT)1是MR 80000的单亚基蛋白，催化核糖核酸酶H(RNaseH)活性(Dickson等人，1982年)。从核苷酸序列中推导出MuLV RT的一级氨基酸序列
Revised Manuscript Received April 10, 1991 abstract: We have labeled theprimer binding domain of murine leukemia virus reverse transcriptase (MuLV RT) by covalently cross-linking 5'end labeled d (T) 8 to MuLV RT, using ultraviolet light energy. The specificity and the functional significance of the primer cross-linking reaction were demonstrated by the fact that (i) other oligomeric primers, tRNAs, and also template-primers readily compete with radiolabeled d (T) 8 for the cross-linking reaction,(ii) under similar conditions, the competing primers and template-primer also inhibit the DNA polymerase activity of MuLV RT to a similar extent,(iii) substrate deoxynucleotides have no effect, and (iv) the reaction is sensitive to high ionic strength. In order to identify the primer binding domains/sites in MuLV RT; tryptic digests prepared from the covalently cross-linked MuLV RT and [32P] d (T) 8 complexes were resolved on C-18 columns by reverse-phase HPLC. Three distinct radiolabeled peptides were found to contain the majority of the boundprimer. Of these, peptide I contained approximately 65% radioactivity, while the remainder was associated with peptides II and III. Amino acid composition and sequence analyses of the individual peptides revealed that peptide I spans amino acidresidues 72-80 in the primary amino acid sequence of MuLV RT and is located in the polymerase domain. The primer cross-linking siteappears to be at or near Pro-76. Peptides II and III span amino acid residues 602-609 and 615-622, respectively, and are located in the RNase H domain. The probable cross-linking sites in peptides II and III are suggested to be at or near Leu-604 and Leu-618, respectively.IV^ oloney murine leukemia virus reverse transcriptase (MuLV RT) 1 is a single-subunit protein of Mr 80000 and catalyzes both RNA-and DNA-directed DNA polymerase activities as well as a ribonuclease H (RNase H) activity (Dickson et al., 1982). The primary amino acidsequence of MuLV RT has been deduced from the nucleotide sequence