Analysis of ColE1 MbeC Unveils an Extended Ribbon-Helix-Helix Family of Nicking Accessory Proteins

ABSTRACT MbeC is a 13-kDa ColE1-encoded protein required for efficient mobilization of ColE1, a plasmid widely used in cloning vector technology. MbeC protein was purified and used for in vitro DNA binding, which showed that it binds specifically double-stranded DNA (dsDNA) containing the ColE1 oriT. Amino acid sequence comparison and secondary structure prediction imply that MbeC is related to the ribbon-helix-helix (RHH) protein family. Alignment with RHH members pointed to a conserved arginine (R13 in MbeC) that was mutated to alanine. The mutant MbeC(R13A) was unable to bind either single-stranded DNA or dsDNA. Limited proteolysis fragmented MbeC in two stable folding domains: the N-terminal domain, which contains the RHH motif, and the C-terminal domain, which comprises a signature shared by nicking accessory proteins. The results indicate that MbeC plays a similar role in conjugation as TraY and TrwA of plasmids F and R388, respectively. Thus, it appears that an extended, possibly universal mechanism of DNA conjugative processing exists, in which oriT-processing is carried out by relaxases assisted by homologous nicking accessory proteins. This mechanism seems to be shared by all major conjugative systems analyzed thus far.

Barley elongation factor 1α: genomic organization, DNA sequence, and phylogenetic implications

A full length cDNA clone encoding the 447 amino acid long barley (Hordeum vulgare cv. Bomi) endosperm elongation factor 1α (eF-1α) was isolated by a differential screening procedure. RFLP mapping of eF-1α showed that the barley genome contains a small eF-1α gene family of 4 copies, with 1 copy of the gene being located on each of chromosomes 2, 4, 6, and 7. Analysis of barley endosperm total proteins by Western blot with antibodies directed towards wheat eF-1α and the sea urchin 51 kDa proteins gave a single band of the expected molecular weight. Amino acid sequence comparison with other plant eF-1α sequences showed that the isolated barley endosperm eF-1α is more similar to the published wheat eF-1α sequence than to eF-1α sequences previously published for the barley cultivars Igri and Dicktoo. The phylogenetic analysis suggests that the barley eF-1α gene family can be divided into two subfamilies and that two ancestral genes existed before the divergence of monocotyledonous and dicotyledonous plants.Key words: endosperm, gene family, RFLP.