Grove A, Lim L
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
J Mol Biol. 2001 Aug 17;311(3):491-502. doi: 10.1006/jmbi.2001.4763.
Prokaryotic genomes are compacted by association with small basic proteins, generating what has been termed bacterial chromatin. The ubiquitous DNA-binding protein HU serves this function. DNA-binding properties of HU from the hyperthermophilic eubacterium Thermotoga maritima are shown here to differ significantly from those characteristic of previously described HU homologs. Electrophoretic mobility shift analyses show that T. maritima HU (TmHU) binds double-stranded DNA with high affinity (K(d)=5.6(+/-0.7) nM for 37 bp DNA). Equivalent affinity is observed between 4 degrees C and 45 degrees C. TmHU has higher affinity for DNA containing a set of 4 nt loops separated by 9 bp (K(d)=1.4(+/-0.3) nM), consistent with its introduction of two DNA kinks. Using DNA probes of varying length, the optimal binding site for TmHU is estimated at 37 bp, in sharp contrast to the 9-10 bp binding site reported for other HU homologs. Alignment of >60 HU sequences demonstrates significant sequence conservation: A DNA-intercalating proline residue is almost universally conserved, and it is preceded by arginine and asparagine in most sequences, generating a highly conserved RNP motif; V substitutes for R only in HU from Thermotoga, Thermus and Deinococcus. A fivefold increase in DNA-binding affinity is observed for TmHU in which V is replaced with R (TmHU-V61R; K(d)=1.1(+/-0.2) nM), but a change in the trajectory of DNA flanking the sites of DNA intercalation is inferred from analysis of TmHU-V61R binding to DNA modified with 4 nt loops or with substitutions of 5-hydroxymethyluracil for thymine. Survival in extreme environments places unique demands on protection of genomic DNA from thermal destabilization and on access of DNA to the cellular machinery, demands that may be fulfilled by the specific DNA-binding properties of HU and by the fine structure of the bacterial chromatin.
原核生物基因组通过与小的碱性蛋白结合而被压缩，从而形成所谓的细菌染色质。普遍存在的DNA结合蛋白HU发挥着这一功能。本文显示，嗜热真细菌海栖热袍菌（Thermotoga maritima）的HU的DNA结合特性与先前描述的HU同源物的特性有显著差异。电泳迁移率变动分析表明，海栖热袍菌HU（TmHU）以高亲和力结合双链DNA（对于37 bp的DNA，K(d)=5.6(±0.7) nM）。在4℃至45℃之间观察到同等亲和力。TmHU对含有由9 bp隔开的一组4 nt环的DNA具有更高的亲和力（K(d)=1.4(±0.3) nM），这与其引入两个DNA扭结一致。使用不同长度的DNA探针，估计TmHU的最佳结合位点为37 bp，这与报道的其他HU同源物的9 - 10 bp结合位点形成鲜明对比。对60多个HU序列的比对显示出显著的序列保守性：一个DNA插入脯氨酸残基几乎普遍保守，并且在大多数序列中它之前是精氨酸和天冬酰胺，形成一个高度保守的RNP基序；仅在热袍菌属、嗜热栖热菌属和异常球菌属的HU中，缬氨酸替代了精氨酸。对于用精氨酸替代缬氨酸的TmHU（TmHU-V61R；K(d)=1.1(±0.2) nM），观察到DNA结合亲和力增加了五倍，但通过分析TmHU-V61R与用4 nt环修饰或用5-羟甲基尿嘧啶替代胸腺嘧啶的DNA的结合，推断出DNA插入位点两侧的DNA轨迹发生了变化。在极端环境中的生存对保护基因组DNA免受热不稳定以及对DNA与细胞机制的接触提出了独特要求，这些要求可能通过HU的特定DNA结合特性和细菌染色质的精细结构来满足。
