TRANSCRIPTION ON NUCLEOSOMAL TEMPLATES BY RNA POLYMERASE-II INVITRO - INHIBITION OF ELONGATION WITH ENHANCEMENT OF SEQUENCE-SPECIFIC PAUSING
TRANSCRIPTION ON NUCLEOSOMAL TEMPLATES BY RNA POLYMERASE-II INVITRO - INHIBITION OF ELONGATION WITH ENHANCEMENT OF SEQUENCE-SPECIFIC PAUSING
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DOI：
10.1101/gad.5.4.683
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发表时间：
1991-04-01
影响因子：
10.5
通讯作者：
LUSE, DS
中科院分区：
文献类型：
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作者：
IZBAN, MG;LUSE, DS
RNA聚合酶II在体内延长RNA链的过程，其中模板至少部分处于核小体构型，仍然知之甚少。为了解决这个问题，我们有部分纯化的RNA聚合酶II转录复合物在延伸早期暂停。然后将这些复合物用作染色质重建的底物。相对于裸DNA模板上的伸长，这些核小体模板上的新生RNA链的伸长受到严重抑制。核小体模板上的延伸导致RNA聚合酶暂停产生的转录物的可再现的模板特异性模式。RNA聚合酶不被终止，因为大多数将在添加Sarkosyl或400 mM KCl后恢复延伸。RNA聚合酶II暂停/终止位点的有效性通过核小体的存在而增强。例如，与c-myc基因外显子1终止子序列相似的暂停位点在重构模板中的使用效率是其4 - 7倍。在具有定相核小体的模板上和在没有显示主要定相模式的重构模板上的伸长的比较表明暂停位点的位置与核小体的位置无关。相反，RNA聚合酶停留在核小体模板上的主要决定因素似乎是潜在的DNA序列。
The process by which RNA polymerase II elongates RNA chains in vivo, where the template is at least partially in a nucleosomal configuration, remains poorly understood. To approach this question we have partially purified RNA polymerase II transcription complexes paused early in elongation. These complexes were then used as substrates for chromatin reconstitution. Elongation of the nascent RNA chains on these nucleosomal templates is severely inhibited relative to elongation on naked DNA templates. Elongation on the nucleosomal templates results in a reproducible template-specific pattern of transcripts generated by RNA polymerase pausing. The RNA polymerases are not terminated because the large majority will resume elongation upon the addition of Sarkosyl or 400 mM KCl. The effectiveness of RNA polymerase II pause/termination sites is enhanced by the presence of nucleosomes. For example, a pause site similar in sequence to the c-myc gene exon 1 terminator is used four to seven times more effectively in reconstituted templates. A comparison of elongation on templates bearing phased nucleosomes and on reconstituted templates that show no predominant phasing pattern indicates that the locations of pause sites are not related to the positions of the nucleosomes. Rather, the major determinant of RNA polymerase pausing on the nucleosomal templates appears to be the underlying DNA sequence.