INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
批准号:
6161965
负责人:
S B ZIMMERMAN
金额:
$0.0万
依托单位国家:
美国
项目类别:
财政年份:
--
资助国家:
美国
项目状态:
未结题
起止时间:
至
中文摘要
大肠杆菌的类核结构在很大程度上是未知的。
作为解决这个长期存在的问题的一种方法,我们广泛地
从大肠杆菌中分离的表征类核制剂
在相对非变性的条件下(亚精胺类核,
Kornberg等人)分离的类核在很大程度上是稳定的
大分子拥挤,与早先提出的In
高浓度环境细菌的体内致密效应
细胞质(印刷中的手稿)。一种相对简单的模式
经消化后,蛋白质从分离的类核中释放出来
胰腺DNA酶。三种小的DNA结合蛋白(HU、H-NS和FIS)
细胞裂解过程中残留的溶菌酶占一半
蛋白质被释放,RNA聚合酶占了大部分
余数。用凝胶电泳法对蛋白质进行鉴定
标准,在H-NS和FIS的情况下,通过它们的N-末端氨基
酸序列。加盐法提取分离的类核物质
在拥挤、稳定的条件下浓缩可以有效地去除
两种重要的蛋白质(Hu和溶菌酶),但形式紧凑
保留了类核的一部分。这些提取的类核物质继续
在重新隔离到最初不拥挤的环境中时,保持其紧凑的状态
盐度适中,说明胡,最常见的是“组蛋白样”。
大肠杆菌的蛋白质,不是维持生命的必要成分
在这些制剂中压实。分离出的类核的暴露
到中等浓度的尿素或氯化钠会导致
如光学显微镜所见,DNA被分解成长长的流光。DNA
与尿素一样,经历了沉降性的特征变化
或者,氯化钠剥离了数量不断增加的类核蛋白。那里
相对于氯化钠的浓度是相当大的滞后
需要去除几种蛋白质,特别是FIS和H-NS,
可能表明存在一个不耐盐的结构,包括
这些蛋白质。我们目前的重点是验证和定义这一点
结构,并与简单地由
这些蛋白质和DNA之间的亲和力。
英文摘要
The structure of the nucleoid of Escherichia coli is largely unknown.
As an approach to this long-standing problem, we have extensively
characterized nucleoid preparations that were isolated from E. coli
under relatively non-denaturing conditions (spermidine nucleoids,
Kornberg et al.) The isolated nucleoids were greatly stabilized by
macromolecular crowding, consistent with earlier suggestions of an in
vivo compacting effect of the highly concentrated, surrounding bacterial
cytoplasm (manuscripts in press). A relatively simple pattern of
proteins was released from the isolated nucleoids upon digestion with
pancreatic DNase. Three small, DNA-binding proteins (HU, H-NS, and Fis)
and residual lysozyme from the cell lysis procedure account for half of
the protein released, with RNA polymerase accounting for much of the
remainder. The proteins have been identified by electrophoretic
criteria and, in the case of H-NS and Fis, by their N-terminal amino
acid sequences. Extraction of isolated nucleoids with elevated salt
concentrations under crowded, stabilizing conditions efficiently removes
two of the prominent proteins (HU and lysozyme), yet the compact form
of the nucleoids is retained. These extracted nucleoids continue to
maintain their compact form upon reisolation into the initial uncrowded
low-salt medium, indicating that HU, the most common "histone-like"
protein of E. coli, is not a necessary component for maintaining
compaction in these preparations. Exposure of the isolated nucleoids
to intermediate concentrations of urea or NaCl results in the unfolding
of the DNA into long streamers, as seen by light microscopy. The DNA
undergoes characteristic changes in sedimentation properties as the urea
or NaCl strips off increasing amounts of the nucleoid proteins. There
is considerable hysteresis with respect to the concentrations of NaCl
required for the removal of several proteins, notably Fis and H-NS,
possibly indicating the existence of a salt-labile structure involving
these proteins. Our current focus is in validating and defining this
structure and distinguishing it from the complexes formed simply due to
the affinity between these proteins and the DNA.
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INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
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批准号:5201983
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负责人:S B ZIMMERMAN
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INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
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批准号:2573111
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INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
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批准号:3854953
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INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
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批准号:3940872
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财政年份:--
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负责人:S B ZIMMERMAN
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INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
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批准号:3776440
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INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
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批准号:3754321
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INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
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批准号:4689922
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INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
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批准号:3876006
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INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
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批准号:3964707
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依托单位:
INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
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批准号:3917964
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依托单位:
INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS
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批准号:3840001
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海外基金