Ogawa J, Long S R
Howard Hughes Medical Institute, Department of Biological Sciences, Stanford University, California 94305-5020, USA.
Genes Dev. 1995 Mar 15;9(6):714-29. doi: 10.1101/gad.9.6.714.
The molecular chaperones related to GroEL (hsp60, cpn60) interact with partially folded proteins and appear to assist them to attain active and correctly folded conformation. They are required for cell viability but are probably more important for some processes than for others. Through a random genetic search to find loci that are required for expression of the Rhizobium meliloti nod (nodulation) genes, we isolated a mutant (B4) defective in luteolin-dependent activation of nod gene expression, and found it carries a Tn5 insertion within a chromosomal groEL gene (groELc) located just downstream of a groESc gene. The groELc mutation affected activity of three related LysR-type activator proteins NodD1, NodD3, and SyrM; on plants, the mutants formed nodules late, and the nodules were Fix-. Hybridization and protein expression analysis show that a similar groESL locus (groESLa) maps to the Rm1021 megaplasmid pSyma. Southern blot analysis revealed additional, but less closely related sequences hybridizing to groELc and groESc probes elsewhere in the R. meliloti genome. Clones of groESLc and groESLa can each restore robust phage lambda growth on an Escherichia coli groE mutant. Likewise each clone can complement all of the phenotypes observed for B4 mutants; thus, the two appear to be functionally equivalent if expression is controlled. We determined that groELc is required for normal DNA binding of the NodD target sequence in R. meliloti. GroEL coimmunopurifies with NodD1 from R. meliloti, which suggests a direct physical association between these proteins. GroEL is thus probably involved in the folding or assembly of transcriptionally active NodD.
与GroEL相关的分子伴侣（热休克蛋白60、伴侣蛋白60）与部分折叠的蛋白质相互作用，似乎有助于它们获得活性且正确折叠的构象。它们是细胞生存所必需的，但可能对某些过程比对其他过程更重要。通过随机基因搜索来寻找苜蓿根瘤菌结瘤（nodulation）基因表达所需的基因座，我们分离出了一个在木犀草素依赖的结瘤基因表达激活方面有缺陷的突变体（B4），并发现它在位于groESc基因下游的染色体groEL基因（groELc）内有一个Tn5插入。groELc突变影响了三种相关的LysR型激活蛋白NodD1、NodD3和SyrM的活性；在植物上，这些突变体结瘤较晚，且结瘤是固氮缺陷型的。杂交和蛋白质表达分析表明，一个相似的groESL基因座（groESLa）定位于Rm1021大质粒pSyma上。Southern印迹分析揭示了在苜蓿根瘤菌基因组其他位置与groELc和groESc探针杂交的其他但相关性较小的序列。groESLc和groESLa的克隆都能在大肠杆菌groE突变体上恢复噬菌体λ的强劲生长。同样，每个克隆都能互补B4突变体所观察到的所有表型；因此，如果表达得到控制，这两者在功能上似乎是等效的。我们确定groELc是苜蓿根瘤菌中NodD靶序列正常DNA结合所必需的。GroEL能与苜蓿根瘤菌中的NodD1共同免疫纯化，这表明这些蛋白质之间存在直接的物理关联。因此，GroEL可能参与了转录活性NodD的折叠或组装。
