斯坦福大学Roge rKornberg因揭示真核转录的分子基础，独得今年诺贝尔化学奖。最近，Kornberg及其同事对真核转录过程中聚合酶Ⅱ（polymerase??II??）的保真性进行了阐释，将研究成果发表在12月1日Cell杂志上。

　　研究人员发现聚合酶II中一个叫做trigger loop的部分，作用如同暗门（trap  door），在与模板匹配的三磷酸腺苷（nucleoside  triphosphate，NTP）下活动，关闭活性中心，与NTP和酶的其它部分组成相互作用的广泛的网络。这些相互作用使trigger  loop的另一条侧链精确定位，以机械地“激活”延长RNA链所需的化学键的形成。如果NTP发生轻微的变动，这些关键的相互作也将失败。

　　trigger  loop机制具有NTP识别和催化双重作用，保证转录的高保真度。Kornberg说从转录复合物结构中得到的信息，也许最为基础。“我们很早就知道酶在选择正确的碱基和糖时精确性很高，但是不知道是怎样实现这种高精确度的。”这些发现提供了“一个意想不到的一流解释，即漂亮又简单，并且被自然一而再地验证了。”

　　转录的基本机制就存在于进化保守的RNA聚合酶中。共同特征包括有15个碱基对的一段未被卷绕的区域。其中与RNA转录物与DNA杂交的八个碱基，位于转录起始位点（transcription  bubble）中心。聚合酶在DNA上可以前后移动，结合NTP后会向前移动，遇到阻碍物后（如受损DNA）后会发生向后运动。

　　2001年，Kornberg小组利用X射线结晶学获得聚合酶II转录复合物的第一张图片，展示了仍有一个核苷酸停留于聚合酶添加位点（addition  site，生物通编者译）的复合物。

　　后来的X射线结果显示，具有添加位点的转录复合物能够纳入相匹配的NTP。这些晶体揭示了聚合酶的第二个NTP结合位点——插入位点（entry  site）。尽管所有的NTP都可结合到插入位点，但是只有与DNA模板相匹配的NTP才能够与添加位点结合，连接到正在延长的RNA链上。酶是利用怎样一种机制精确区分匹配和错误匹配的NTP的至今仍没有答案。

　　RNA碱基（腺嘌呤、胞嘧啶、鸟嘌呤和尿嘧啶）与其互补的DNA模板上的碱基之间的化学吸引，来自于聚合酶Ⅱ的难以置信的选择性。但是研究人员不清楚的是聚合酶是怎样避免把构成DNA链的NTP添加到RNA链上的，毕竟脱氧核核苷酸和核糖核苷酸只是相差一个氧原子。

　　此次实验中，研究人员检测了数百个晶体，获得比前次更高清晰的图片和高质量数据。Kornberg说此次实验将目光放在先前从未重视的一些现象——在配对碱基之下（beneath）的附加蛋白密度（protein??density，生物同编者译）。追溯这种蛋白密度一直到达聚合酶II的trigger  loop区域。

　　Kornberg说现在报道的14种观察到有trigger  loop的晶体结构，只有两种的trigger  loop位于NTP下方。也只有这两种晶体中，NTP能与DNA模板精确配对，证明trigger  loop“与选择NTP有明显关系。”

　　进一步研究发现，当一个匹配的NTP到达添加位点后，trigger  loop在其未知较远的通常所在的位点摆动，直至与NTP平行，然后与NTP的组成部分形成一个相互作用的网络，这个过程“识别添加位点的NTP的所有特征，检测其精确定位。”

　　Kornberg 说：“这种特异性是与NTP平行排列的结果，依赖于碱基、糖、磷酸和trigger  loop摆动的位置，假如偏离，即便很小，这种相互接触也不会发生。”作为平行排列的结果，一亿分之一厘米（埃）的精度。

　　Kornberg说，为了NTP能够通过形成磷酸二酯键（phosphodiester  bond）连接到RNA链上，trigger  loop停留在β磷酸的一个组胺酸侧链的键必须断裂，提示侧有对于键的形成有刺激作用。整个的决策过程极为迅速（皮秒级别）。

　　研究人员总结到：“RNA聚合酶转录DNA的高度特异性的基础在于一个名为trigger  loop的结构，其与聚合酶活性中心的核苷酸的所有方面进行直接或者间接的接触。”

英文原文：
Nobel Laureate Finds 'Elegant' Explanation For DNA Transcribing Enzyme's High Fidelity
Last month, Roger Kornberg of Stanford University won the Nobel Prize in Chemistry for his efforts to unravel the molecular basis of eukaryotic transcription, in which enzymes give 'voice' to DNA by copying it into the RNA molecules that serve as templates for protein in organisms from yeast to humans. Now, Kornberg and his colleagues report in the December 1, 2006 issue of the journal Cell, published by Cell Press, new structures that reveal another critical piece of the puzzle: how the so-called polymerase II enzyme discriminates among potential RNA building blocks to ensure the characteristic accuracy of the process.

The researchers found that a portion of the enzyme known as the trigger loop acts like a 'trap door,' swinging beneath a matching nucleoside triphosphate (NTP) building block, to close off the active center and form an extensive network of interactions with the NTP and other parts of the enzyme. Those interactions leave another side chain in the trigger loop precisely positioned, such that it may literally 'trigger' the formation of the chemical bonds that link components of the growing RNA chain together. If the NTP is even slightly misaligned, Kornberg said, those critical interactions fail.

The trigger loop mechanism therefore couples NTP recognition and catalysis, ensuring the fidelity of transcription, they reported.

"Of all revelations from the structure [of the transcription machinery] since it was first solved, this is perhaps the most fundamental since it gets at the underlying mechanisms," Kornberg said. "It's long known that the enzyme operates with high fidelity - selecting the correct base and sugar - but it's been a mystery how that is accomplished."

These findings offer "an unexpected and elegant explanation that's both beautiful and simple, as nature invariably proves to be."

The fundamental mechanism of transcription is conserved among cellular RNA polymerases, the researchers explained. Common features include an unwound region of about 15 base pairs of the DNA with some eight residues of the RNA transcript hybridized with the DNA in the center of the 'transcription bubble.' The enzyme polymerases involved are capable of moving both forward and backward on the DNA. Forward movement is favored by the binding of NTPs, while backtracking occurs especially when the enzyme encounters an impediment, such as damaged DNA.

Kornberg's group captured the first picture of the polymerase II transcribing complex by X-ray crystallography in 2001. Those structures revealed the complex with a nucleotide still in the enzyme's addition site, just after it had been added to the RNA transcript.

Later X-ray structures revealed the transcribing complex with the addition site available for entry of a matched NTP. Those crystals uncovered a second NTP-binding site on the transcribing enzyme, dubbed the entry site. While all NTPs can bind the entry site, only an NTP matched for base-pairing with the DNA template binds the addition site for attachment to the growing RNA strand, Kornberg said.

Yet the question of how the enzyme achieves such a high degree of discrimination between matched and mismatched NTPs remained unanswered.

The chemical attraction alone between RNA bases - adenine, cytosine, guanine, and uracil - and their complementary bases on the DNA template strand is far from sufficient to account for the incredible selectivity of polymerase II, Kornberg said. And the scientists didn't know either how the polymerase avoids substituting the NTPs that constitute DNA for the correct RNA building blocks, molecules that differ by only one oxygen atom.

In search of an explanation in the current study, the researchers screened hundreds of crystals to achieve higher data quality and resolution than ever before.

"In the course of the work, we saw something that had never been noticed before - additional protein density beneath the matching nucleotide," Kornberg said.

The team traced that protein density back to a portion of the polymerase II enzyme: the trigger loop.

"Of the 14 crystal structures now reported in which the trigger loop was observed, only in two is it seen in that location, directly beneath the NTP," Kornberg said. "Those were the only two crystals in which the NTP was correctly matched to the DNA template, evidence of the trigger loop's clear relationship to NTP selection."

Further study revealed that, when a matching NTP reaches the addition site, the trigger loop swings from its usual position some distance away until it rests parallel to the NTP. It then forms a network of interactions - some 20 to 30 in all - with components of the NTP, a process that serves to "recognize all features of the NTP in the addition site and detect its precise location," the researchers reported.

"The specificity is a result of the alignment with the NTP that is critically dependent upon the base, sugar, phosphate and location when the trigger loop swings into position," Kornberg said. "If it is misaligned even slightly, that set of contacts cannot occur."

As a consequence of that alignment, to angstrom (a unit of length equal to one hundred millionth of a centimeter) precision, a histidine side chain of the trigger loop rests on the phosphate, the chemical constituent that must have its bond broken in order for the NTP to join the RNA chain through the formation of a phosphodiester bond, Kornberg said. The finding suggested the side chain acts as a trigger for bond formation.

The whole decision-making process occurs extremely rapidly, he added, on the order of picoseconds. A picosecond is one trillionth of a second.

"The basis for the extraordinary specificity with which RNA polymerases transcribe DNA lies in a structural element termed the trigger loop, which makes both direct and indirect contact with all features of the nucleotide in the polymerase active center," the researchers concluded.

                                                                               摘自《生物谷》