Positive Control Mechanism Of Rhizobium NodD Operator

In Rhizobium leguminosarum, most nodulation genes (nod, noe and nol)participate in the synthesis or secretion of a family of lipochito-oligosaccharidemolecules, the Nod factors, which are required for bacterial entry into root hairs. Inthe perspective of transcriptional control, nod genes exist as a whole NodD regulon,which includes multiple NodD operons. Different NodD operons are highlyconserved in their promoter DNA sequences. NodD, member of the prokaryoticLysR family transcriptional factors, can positively activate the transcription ofinducible nod genes in response to signal molecules flavonoids from host plants.Insight into the molecular mechanism of NodD operator not only helps to understandthe signal exchange process in detail but also contributes to revealing thetranscriptional regulation mechanism controlled by the LysR family, the largesttranscriptional factor family in prokaryotes.We have chosen Rhizobium leguminosarum nodA-nodD promoter as researchtarget. Based on our data, we have proposed a NodD-mediated positive controlmodel. NodD binds to and bends target promoters through anchoring two tandem andindividual specific DNA sites. NodD functions as a tetramer, which has a V-shapedmain body. Tetrameric NodD is to change its own conformation rather than its - v -中国科学院上海生物化学和细胞生物学研究所博士论文 摘 要oligomeric forms in response to small signal molecules. The specific interactionbetween each NodD DNA-binding domain and each specific DNA site does not alteritself in spite of naringenin induction, and the induced conformational change istransferred from protein to DNA. Only the DNA conformation incited by inducedNodD is competent for RNA polymerase to form the transcriptional open complex. Itcannot be excluded that NodD may have protein-to-protein contacts with RNApolymerase, and that the NodD conformational change may also directly contributeto the transcriptional open complex formation. However, the NodD conformationalchange itself cannot serve as the determinant of the transcriptional molecular switch.Besides, two proteins, Rhizobium HU-like protein Hurl and iron mechanism-relevant sigma factor RpoI, have been preliminarily studied. Hurl is found to bindspecifically to nodA, nodF and nodM promoters. Once binding to target DNA, Hurlcan affect the interaction between its neighboring proteins and their correspondingDNA targets. Thus, it may functionally play a role of assisting NodD to integrate thewhole NodD regulon. We have demonstrated that the renatured Escherichia coli-overexpressed RpoI can reconstitute with Rhizobium core RNA polymerase in vitro.A preparation procedure has been established, which is expected to be useful tofurther functional study.