Characterization of RNA-binding properties of three types of RNA-binding proteins in Anabaena sp. PPC 7120.

The Rbp proteins in cyanobacteria are RNA-binding proteins with a single RNA recognition motif or RRM. A comprehensive assembly of genomic data suggests that there are two major classes of Rbp proteins (classes I and II) that diverged before the diversification of cyanobacteria. Class I proteins are further classified into two types with or without a C-terminal glycine-rich domain. The results of selection from a random RNA pool suggest that RbpA1 (class I) has affinity to C-rich and G-rich sequences. In vitro RNA binding assay with homopolymers indicated that class II protein has low affinity to poly(G) in contrast with class I proteins. Site-specific mutagenesis analysis of the RRM in RbpA1 showed that the aromatic residues Tyr4 or Phe46 are important in RNA binding as well as maintenance of secondary structure. We also tested various truncated proteins lacking the C-terminal domain as well as point mutants. Most of these proteins exhibited decreased affinity to RNA. Circular dichroism analysis as well as chromatographic analysis showed that Tyr4 and Phe46 are also important in maintaining the structure of RbpA1 protein. The C-terminal glycine-rich domain itself does not contribute much to the RNA-binding, but Arg83 which is located close to the C-terminal end of RRM is important in the RNA-binding.

Characterization of genes encoding multi-domain proteins in the genome of the filamentous nitrogen-fixing Cyanobacterium anabaena sp. strain PCC 7120.

Computational analysis of gene structures in the genome of Anabaena sp. PCC 7120 revealed the presence of a large number of genes encoding proteins with multiple functional domains. This was most evident in the genes for signal transduction pathway and the related systems. Comparison of the putative amino acid sequences of the gene products with those in the Pfam database indicated that and PAS domains which may be involved in signal recognition were extremely abundant in Anabaena: 87 GAF domains in 62 ORFs and 140 PAS domains in 59 ORFs. As for the two-component signal transduction system, 73, 53, and 77 genes for simple sensory His kinases, hybrid His kinases and simple response regulators, respectively, many of which contained additional domains of diverse functions, were presumptively assigned. A total of 52 ORFs encoding putative Hanks-type Ser/Thr protein kinases with various domains such as WD-repeat, GAF and His kinase domains, as well as genes for presumptive protein phosphatases, were also identified. In addition, genes for putative transcription factors and for proteins in the cAMP signal transduction system harbored complex gene structures with multiple domains.

Inhibition of the acid lipase activity by apolipoprotein A-I in the presence of lysosomal proteases.

It was found that the inhibition of the lysosomal acid lipase activity by rat apolipoprotein A-I (apo A-I) was increased with the degradation of apo A-I by the lysosomal proteases. We demonstrated that apo A-I could effectively inhibit the acid lipase activity even in the presence of the lysosomal proteases using the hepatic lysosomal fraction.