细胞核与染色质--学生打印版

1、细胞核与染色质细胞核与染色质 王飞 内容提要内容提要 基本结构及主要功能元件1 核内分子生物学过程概述2 核孔复合体及其介导的物质运输3 核膜的崩解与组装4 核仁及其他核内结构的组成与功能5 细胞核基本结构细胞核基本结构 v核被膜 核基质 染色质 核仁 核体 核被膜的结构组成核被膜的结构组成 核被膜 (nuclear envelope) 核膜(nuclear membrane) 核孔复合体(nuclear pore complex,NPC) 核纤层(nuclear lamina) 核膜核膜(nuclear membrane) v外核膜 核糖体颗粒 与内质网相连 v内核膜 无核糖体颗粒 LBR

2、v核周间隙 与内质网腔相通 v核孔 内外膜融合 孔膜区 核孔复合体核孔复合体(NPC) v孔膜区域组装的复杂蛋白复合物 核质物质运输 v核纤层蛋白(lamin A-C) 纤维状网络结构 v结构支撑 调节基因表达 调节DNA损伤修复 v内核膜 NPC 核周染色质 核纤层核纤层(nuclear lamina) v分裂期 核被膜的崩解与组装 核基质核基质 v核骨架 v蛋白质纤维网架结构体系 v真核生物细胞核起源于原核细胞入侵？融合？ v细胞生物学：不同染色质形态的核内分布 核周异染色质 袢环结构(loop) 染色体区域化分布 染色质染色质 核仁核仁 v 核仁：染色体的核仁组织区 RNA 蛋白 核糖体

3、工厂 v 核体：功能元件 RNP集中区域 非编码RNA 蛋白 RNA修饰 RNP组装 核糖体装配 核体核体 核内分子生物学过程核内分子生物学过程 v染色质结构 调节 vDNA复制、 修复、重组 v基因转录及 其调控 vRNA 加工 短片：短片：chromosome coil In this animation well see the way our DNA is tightly packed up to fit into the nucleus of every cell. The process starts with assembly of a nucleosome, which is

4、formed when eight separate histone protein subunits attach to the DNA molecule. The combined tight loop of DNA and protein is the nucleosome. Six nucleosomes are coiled together and these then stack on top of each other. The end result is a fiber of packed nucleosomes known as chromatin. 短片：短片：DNA r

5、eplication Using computer animation based on molecular research, we are able to picture how DNA is replicated in living cells. You are looking at an assembly line of amazing miniature biochemical machines that are pulling apart the DNA double helix and cranking out a copy of each strand. The DNA to

6、be copied enters the production line from bottom left. The whirling blue molecular machine is called a helicase. It spins the DNA as fast as a jet engine as it unwinds the double helix into two strands. One strand is copied continuously and can be seen spooling off to the right. Things are not so si

7、mple for the other strand because it must be copied backwards. It is drawn out repeatedly in loops, and copied one section at a time. The end result is two new DNA molecules. 短片：短片：strand exchange Cross-strand exchange is an important intermediate structure in general DNA recombination. The characte

8、ristic junction is formed when two homologous double-stranded DNA molecules reciprocally exchange DNA strands. This junction can be visualized directly in the electron microscope. An important aspect of recombination is that the junction point is not fixed after it has formed, but can migrate up and

9、 down the DNA as shown in this animation. In the cell, this junction is formed and stabilized by a specific protein, which can be seen here in the background. 短片：短片：gene transcription Transcription is the process by which DNA is copied into RNA in the first step of gene expression. It begins with a

10、bundle of factors assembling at the start of a gene, that is, a linear sequence of DNA instructions, here shown stretching away to the left. The assembled factors include an RNA polymerase, the blue molecule. Suddenly, RNA polymerase is let go, racing along the DNA to read the gene. As it unzips the

11、 double helix, it copies one of the two strands. The yellow chain snaking out of the top is the RNA, a copy of the genetic message. The nucleotide building blocks that are used to make the RNA enter through an intake hole in the polymerase. In the active site of the enzyme, they are then matched to

12、the DNA, nucleotide by nucleotide, to copy the As, Cs, Ts and Gs of the gene. The only difference is that in the RNA copy, thymine is replaced with the closely related base uracil, commonly abbreviated “U”. You are watching this process, called transcription, in real time. 核孔复合体核孔复合体-核质转运通道核质转运通道 v跨

13、内外核膜 亲水通道 v核孔直径 80-120nm 复合体直径120-150nm v自由扩散 顺浓度梯度 有效直径 9-10nm 40-60KD 无需能量 v主动运输 信号肽 需能量 不去折叠 双向同时运输 转运速度极快（500个/s） 短片：短片：Rapid nuclear importation Nuclear import and export can be directly visualized in living cells that express the green fluorescent protein GFP fused to the gene regulatory prote

14、in NF-AT. NF-AT is normally localized in the cytosol, and excluded from the nucleus. But when the cytosolic calcium concentration is raised, NF-AT migrates to the nucleus. This is done here experimentally by adding an ionophore that allows calcium to enter the cells from the medium. Upon removal of

15、the ionophore, calcium levels return to normal and NF-AT is exported from the nucleus. Readdition of the ionophore triggers reimport of NF-AT. 核孔复合体的结构核孔复合体的结构 v30余种蛋白亚基 多拷贝组合 八边形对称结构 柱状亚基 环带亚基 腔内亚基 环状亚基 蛋白纤维 核篮 核孔复合体的结构核孔复合体的结构 核孔复合体转运的分子尺寸限制核孔复合体转运的分子尺寸限制 v无定形结构域(unstructured region) 无序屏障 主动运输与信号肽

16、主动运输与信号肽 v 蛋白分选：信号肽 核转运信号(NTS) v 核分选：核定位信号(NLS) 核输出信号(NES) 任何位置 完成分选后不剪切 核孔复合体转运的生物大分子核孔复合体转运的生物大分子 vNTS/向导蛋白 v转入(Import) 组蛋白 DNA聚合酶 RNA聚合酶 RNA加工相关蛋白 v转出(Export) tRNA mRNA 核糖体前体颗粒 v转入&转出 基因转录调控蛋白（转录因子） RNA向导蛋白 主动运输模型主动运输模型 v通道：NPC 出入口：蛋白纤维 v搬运工：核转运受体 (nuclear transport receptor) v辅助：FG-repeats Ran G

17、TP v 两类核转运受体(NTR) 入核转运受体(NIR) 识别NLS 蛋白入核 出核转运受体(NER) 识别NES 蛋白出核 v 直接结合：多种NTR 识别不同NTS v 间接结合：适配蛋白(adaptor protein) v 无信号肽大分子 “搭车”：RNA 蛋白质 核转运受体对靶蛋白的识别核转运受体对靶蛋白的识别 GTPase：Ran v导航： 控制转运方 向 v调控因子： Ran-GAP Ran-GEF Ran控制的控制的NTR分子开关分子开关 NPC主动运输的分子模型主动运输的分子模型 RNA的出核转运的出核转运 v 向导分子：具有NES的辅助蛋白 v 质量检查：正确折叠和加工的R

18、NA分子才能 获得出核资格 核膜的崩解与组装核膜的崩解与组装-骨架章交叉骨架章交叉 v发生于细胞分裂过程中 v触发点：磷酸化 p34cdc2 N端Ser22 C端Ser392 v Lamin A 单体形式游离 Lamin B 与核膜小泡结合 核膜的崩解与组装核膜的崩解与组装 核膜的崩解与组装核膜的崩解与组装 1 核孔复合体和核被膜片段如何找到染色体？ 2 游离到胞质中的核蛋白如何回到核内？ 3 如何避免不属于核内的胞质蛋白在核膜组装 时混入核内？ 找到染色体找到染色体 v前期核膜崩解后 游离于胞质内： 核膜小泡(双层核膜+膜蛋白) NPC亚基蛋白+NIR FG repeats Lamin Ran Ran-GAP v 末期染色体上： Ran-GEF Ran-GTP Cloud vLamin去磷酸化启动核膜重组： 核膜小泡 Lamin NPC蛋白+NIR NPC+NIR Ran-GTP Cloud 核蛋白回归及非核蛋白屏蔽核蛋白回归及