A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, while loss of the novel HBD led to an ∼90% drop in Kcat with a decreased KM, and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite-binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo cross-linking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, we were not able to document direct and stable interactions with mtSSB when the proteins were co-overexpressed in S2 cells, and functional interactions between them in vitro were minor.

The AtMYB12 activation domain maps to a short C-terminal region of the transcription factor

TheArabidopsis thalianaR2R3-MYB transcription factor MYB12 is a light-inducible, flavonol-specific activator of flavonoid biosynthesis. The transactivation activity of theAtMYB12 protein was analyzed using a C-terminal deletion series in a transientA. thalianaprotoplast assay with the goal of mapping the activation domain (AD). Although the deletion of the last 46 C-terminal amino acids did not affect the activation capacity, the deletion of the last 98 amino acids almost totally abolished transactivation of two different target promoters. A domain swap experiment using the yeast GAL4 DNA-binding domain revealed that the region from positions 282 to 328 ofAtMYB12 was sufficient for transactivation. In contrast to the R2R3-MYB ADs known thus far, that ofAtMYB12 is not located at the rearmost C-terminal end of the protein. TheAtMYB12 AD is conserved in other experimentally proven R2R3-MYB flavonol regulators from different species.

Abi Is Required for Modulation and Stability but Not Localization or Activation of the SCAR/WAVE Complex

ABSTRACT The SCAR/WAVE complex drives actin-based protrusion, cell migration, and cell separation during cytokinesis. However, the contribution of the individual complex members to the activity of the whole remains a mystery. This is primarily because complex members depend on one another for stability, which limits the scope for experimental manipulation. Several studies suggest that Abi, a relatively small complex member, connects signaling to SCAR/WAVE complex localization and activation through its polyproline C-terminal tail. We generated a deletion series of the Dictyostelium discoideum Abi to investigate its exact role in regulation of the SCAR complex and identified a minimal fragment that would stabilize the complex. Surprisingly, loss of either the N terminus of Abi or the C-terminal polyproline tail conferred no detectable defect in complex recruitment to the leading edge or the formation of pseudopods. A fragment containing approximately 20% Abi—and none of the sites that couple to known signaling pathways—allowed the SCAR complex to function with normal localization and kinetics. However, expression of N-terminal Abi deletions exacerbated the cytokinesis defect of the Dictyostelium abi mutant, which was earlier shown to be caused by the inappropriate activation of SCAR. This demonstrates, unexpectedly, that Abi does not mediate the SCAR complex's ability to make pseudopods, beyond its role in complex stability. Instead, we propose that Abi has a modulatory role when the SCAR complex is activated through other mechanisms.

C terminus of NisI provides specificity to nisin

Nisin-producing Lactococcus lactis protects its own cell membrane against the bacteriocin with the ABC transporter NisFEG, and the immunity lipoprotein NisI. In this study, in order to localize a site for specific nisin interaction in NisI, a C-terminal deletion series of NisI was constructed, and the C-terminally truncated NisI proteins were expressed in L. lactis. The shortest deletion (5 aa) decreased the nisin immunity capacity considerably in the nisin-negative strain MG1614, resulting in approximately 78 % loss of immunity function compared with native NisI. A deletion of 21 aa decreased the immunity level even more, but longer deletions, up to 74 aa, provided the same level of nisin immunity as the 21 aa deletion, i.e. approximately 14 % of the immunity provided by native NisI. Similar to native NisI, all the C-terminally truncated NisI proteins provided higher immunity to nisin in the NisFEG-expressing strain NZ9840 than in MG1614, i.e. approximately 40–50 % of the immunity capacity of native NisI. Then, it was determined whether the NisI C-terminal 21 aa fragment could protect cells against nisin. To target the 21 aa fragment to its natural location, 21 C-terminal amino acids from the subtilin-specific immunity lipoprotein SpaI were replaced by 21 C-terminal amino acids from NisI. The expression of the SpaI′–′NisI fusion in L. lactis strains significantly increased their nisin immunity. This is the first time the immunity function of a lantibiotic immunity protein has been transferred to another protein. However, unlike native NisI, and the C-terminally truncated NisI fragments, the increase in nisin immunity conferred by the SpaI′–′NisI fusion was the same in both the NisFEG strain NZ9840 and MG1614. In conclusion, the SpaI′–′NisI fusion could not enhance nisin immunity by interacting with NisFEG, whereas the C-terminally truncated NisI fragments and native NisI were able to enhance nisin immunity, probably by co-operation with NisFEG. The results made it evident that the C terminus of NisI is involved in specific interaction with nisin, and that it confers specificity for the NisI immunity lipoprotein.

The Exon 6ABC Region of Amelogenin mRNA Contribute to Increased Levels of Amelogenin mRNA through Amelogenin Protein-enhanced mRNA Stabilization

We recently demonstrated that the reuptake of full-length amelogenin protein results in increased levels of amelogenin mRNA through enhanced mRNA stabilization (Xu, L., Harada, H., Tamaki, T. Y., Matsumoto, S., Tanaka, J., and Taniguchi, A. (2006) J. Biol. Chem. 281, 2257–2262). Here, we examined the molecular mechanism of enhanced amelogenin mRNA stabilization. To identify the cis-regulatory region within amelogenin mRNA, we tested various reporter systems using a deletion series of reporter plasmids. A deletion at exon 6ABC of amelogenin mRNA resulted in a 2.5-fold increase in the amelogenin mRNA expression level when compared with that of full-length mRNA, indicating that a cis-element exists in exon 6ABC of amelogenin mRNA. Furthermore, Northwestern analysis demonstrated that amelogenin protein binds directly to its mRNA in vitro, suggesting that amelogenin protein acts as a trans-acting protein that specifically binds to this cis-element. Moreover, recombinant mouse amelogenin protein extended the half-life of full-length amelogenin mRNA but did not significantly alter the half-life of exon 6ABC-deletion mutant mRNA. The splice products produced by deletion of exon 6ABC are known as leucine-rich amelogenin peptides and have signaling effects on cells. Our findings also suggest that the regulation of full-length amelogenin protein expression differs from the regulation of leucine-rich amelogenin peptide expression.

A Promoter from Pea Gene DRR206 Is Suitable to Regulate an Elicitor-Coding Gene and Develop Disease Resistance

Plant nonhost disease resistance is characterized by the induction of multiple defense genes. The pea DRR206 gene is induced following inoculation with pathogens and treatment with abiotic agents, and moderately induced by wounding. A deletion series of DRR206 promoter segments was fused with the β-glucuronidase (GUS) reporter gene and transiently transferred to tobacco, potato, and pea. GUS activity revealed that two upstream regions of the DRR206 promoter were particularly important for activation in the three plant species. Putative cis regulatory elements within the DRR206 promoter included a wound/pathogen- inducible box (W/P-box) and a WRKY box (W-box). Gel shift assays with nuclear extracts from treated and untreated tissue with the W/P-box revealed both similar and unique protein-DNA complexes from pea, potato, and tobacco. Tobacco was stably transformed with gene constructs of the DRR206 promoter fused with a DNase elicitor gene from Fusarium solani f. sp. phaseoli, FsphDNase. Pathogenicity tests indicated that the FsphDNase elicitor conferred resistance against Pseudomonas syringae pv. tabaci and Alternaria alternata in tobacco. Transgenic potatoes showed some sensitivity to the FsphDNase gene providing less protection against Phytophthora infestans. Thus, the elicitor-coding gene, FsphDNase, is capable of generating resistance in a heterologous plant system (tobacco) when fused with defined regions of the pea DRR206 promoter.

A transient assay for evaluating promoters in wheat endosperm tissue

A transient assay was developed for the evaluation of promoter sequences in wheat endosperm tissue. A deletion series from an omega-secalin gene promoter, located on chromosome 1RS.1DL of specific wheat lines, were translationally fused to a uidA reporter gene. These promoters were evaluated for expression in wheat endosperm tissue after integration of the DNA into the cell using microprojectile bombardment. The results were compared with those obtained using other transient assay systems.Key words: particle bombardment, transient assay, omega-secalin gene, wheat endosperm.

Core binding factor cannot synergistically activate the myeloperoxidase proximal enhancer in immature myeloid cells without c-Myb.

The myeloperoxidase (MPO) gene is transcribed specifically in immature myeloid cells and is regulated in part by a 414-bp proximal enhancer. Mutation of a core binding factor (CBF)-binding site at -288 decreased enhancer activity 30-fold in 32D cl3 myeloid cells cultured in granulocyte colony-stimulating factor (G-CSF). A novel functional analysis, linking the CBF-binding site to an enhancer deletion series, located at -147 an evolutionarily conserved c-Myb-binding site which was required for optimal enhancer activity and synergy with CBF in 32D cells. These sites cooperated in isolation and independent of a precise spacing. Deletional analysis carried out in the absence of the c-Myb-binding site at -147 located at -301 a second c-Myb-binding site which also synergized with CBF to activate the enhancer. A GA-rich region at -162 contributed to cooperation with CBF when the adjacent c-Myb-binding site was intact. Mutation of both c-Myb-binding sites in the context of the entire enhancer greatly impaired activation by endogenous CBF in 32D cells. Similarly, activation by c-Myb was impaired in constructs lacking the CBF-binding site. CBF and c-Myb were required for induction of MPO proximal enhancer activity when 32D cells differentiated in response to G-CSF. A fusion protein containing the Gal4 DNA-binding domain and the AML-1B activation domain, amino acids 216 to 480, activated transcription alone and cooperatively with c-Myb in nonmyeloid CV-1 cells. Determining how CBF and c-Myb synergize in myeloid cells might contribute to our understanding of leukemogenesis by the AML1-ETO, AML1-MDS1, CBFbeta-SMMHC, and v-Myb oncoproteins.