Deng L, Shuman S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Biochemistry. 1997 Dec 16;36(50):15892-9. doi: 10.1021/bi972037a.
We have analyzed the elongation properties of vaccinia virus RNA polymerase during a single round of transcription in vitro. RNA-labeled ternary complexes were halted at a unique template position located upstream of a T-run (TTTTTTTTT) in the nontemplate strand; this element encodes an RNA signal for factor-dependent transcription termination at distal sites on the template. The halted ternary complexes were purified and allowed to resume elongation under a variety of conditions. We found that the T-run constituted a strong elongation block, even at high nucleotide concentrations. The principal sites of pausing were at a C position situated two nucleotides upstream of the first T in the T-run and at the first three to four T positions within the T-run. There was relatively little pausing at the five downstream Ts. Intrinsic pausing was exacerbated at suboptimal nucleotide concentrations. Ternary complexes arrested by the T-run at 10 microM NTPs rapidly traversed the T-run when the NTP pool was increased to 1 mM. Limiting GTP (1 microM) resulted in polymerase stuttering at the 3' margin of the T-run, immediately prior to a templated G position; this generated a ladder of slippage synthesis products. We found that vaccinia ternary complexes remained intact after elongating to the very end of a linear DNA template and that such complexes catalyzed the addition of extra nucleotides to the 3' end of the RNA chain. The 3' end addition required much higher concentrations of NTPs than did templated chain elongation. Finally, we report that factor-dependent transcription termination by vaccinia RNA polymerase downstream of the T-run was affected by nucleotide concentration. Limiting UTP caused the polymerase to terminate at sites closer to the UUUUUNU termination signal. This is consistent with the kinetic coupling model for factor-dependent termination.
我们分析了痘苗病毒RNA聚合酶在体外单轮转录过程中的延伸特性。用RNA标记的三元复合物在非模板链上T串（TTTTTTTTT）上游的一个独特模板位置处停滞；该元件编码一个RNA信号，用于模板远端位点的因子依赖性转录终止。将停滞的三元复合物纯化，并在各种条件下使其恢复延伸。我们发现，即使在高核苷酸浓度下，T串也构成了一个强大的延伸障碍。主要的暂停位点位于T串中第一个T上游两个核苷酸处的C位置以及T串内的前三个至四个T位置。在下游的五个T处暂停相对较少。在次优核苷酸浓度下，内在暂停会加剧。当NTP池增加到1 mM时，在10 μM NTPs下被T串阻滞的三元复合物会迅速穿过T串。限制GTP（1 μM）会导致聚合酶在T串的3'边缘、紧接模板G位置之前发生口吃；这产生了一系列滑移合成产物。我们发现痘苗三元复合物在延伸至线性DNA模板的末端后仍保持完整，并且这样的复合物催化在RNA链的3'末端添加额外的核苷酸。3'末端添加所需的NTP浓度比模板链延伸所需的浓度高得多。最后，我们报告痘苗RNA聚合酶在T串下游的因子依赖性转录终止受核苷酸浓度影响。限制UTP会导致聚合酶在更接近UUUUUNU终止信号的位点终止。这与因子依赖性终止的动力学偶联模型一致。
