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.

Studies on the molecular mechanism of the interactions between the cyanobacterial transcription factor, SmtB, and its recognition DNA sequences.

Tolerance to the heavy-metal ion stress in cyanobacterial cell is regulated by the transcriptional repressor SmtB that senses the Zn/Cd concentration in the cell. SmtB regulated the transcriptional level of class II metallothionein, SmtA. There are two recognition DNA sequences (Bbs1 and Bbs2) in the operator/promoter region of smtA gene. To clear the functional meaning of the presence of these two sequences, we have compared the affinities of native and point-mutated SmtBs to these two sites. We also have compared the sizes of the protein-DNA complexes being formed with these two sites. SmtB forms protein-DNA complex in an unique size with Bbs1, in three different sizes with Bbs2. We have further designed the modified Bbs1 and Bbs2 sequences, and the results obtained with these sequences indicate that the differences observed between the cases of Bbs1 and Bbs2 are caused by the presence of direct repeat sequence and the differences in the linker sequences.

Physicochemical analysis of the interaction between a metal ion and the metal-ion-binding motif in hammerhead ribozymes.

Hammerhead ribozymes have a metal-ion-binding motif in the conserved core region, and this motif can capture several kinds of metal ions. In order to determine what kind of metal ions can be recognized by the motif, we measured 2D 1H-1H NOESY spectra in the presence of various metal ions, such as Cd2+, Na+ and Co(NH3)6(3+) ions, as well as in the absence of these ions. We have completed the assignment of the resonances of the base protons and anomeric protons in all cases, and determined their chemical shift perturbations.

FILAMENTOUS FLOWER, a meristem and organ identity gene of Arabidopsis, encodes a protein with a zinc finger and HMG-related domains.

Distinctive from that of the animal system, the basic plan of the plant body is the continuous formation of a structural unit, composed of a stem with a meristem at the top and lateral organs continuously forming at the meristem. Therefore, mechanisms controlling the formation, maintenance, and development of a meristem will be a key to understanding the body plan of higher plants. Genetic analyses of filamentous flower (fil) mutants have indicated that FIL is required for the maintenance and growth of inflorescence and floral meristems, and of floral organs of Arabidopsis thaliana. FIL encodes a protein carrying a zinc finger and a HMG box-like domain, which is known to work as a transcription regulator. As expected, the FIL protein was shown to have a nuclear location. In situ hybridization clearly demonstrated that FIL is expressed only at the abaxial side of primordia of leaves and floral organs. Transgenic plants, ectopically expressing FIL, formed filament-like leaves with randomly arranged cells at the leaf margin. Our results indicate that cells at the abaxial side of the lateral organs are responsible for the normal development of the organs as well as for maintaining the activity of meristems.

Implications of the zinc-finger motif found in the DNA-binding domain of the human XPA protein.

BACKGROUND: The XPA (xeroderma pigmentosum group A) protein specifically recognizes the UV-or chemically damaged DNA lesions, and triggers the nucleotide excision repair process. This XPA protein contains the functional domain which is crucial to the recognition of damaged DNA. Its primary structure suggests that this DNA binding domain may contain a zinc-finger motif. To gain a more detailed insight into this zinc-finger motif, we have measured the 113Cd-NMR spectra of the DNA binding domains derived from the wild-type and mutant XPA proteins. RESULTS: 113Cd-NMR analysis, combined with atomic absorption and site-directed mutagenesis, revealed that the DNA binding domain contains one zinc ion, coordinated with four cysteine residues (Cys105, Cys108, Cys126, and Cys129), that is essential for correct protein folding in vivo and in vitro. CONCLUSIONS: The four ligand cysteine residues form a Cys-X2-Cys-X17-Cys-X2-Cys motif, which is reminiscent of the (Cys)4 type zinc-finger motif found in numerous transcriptional regulatory proteins. However, the secondary structure prediction and the 3D-1D compatibility analysis demonstrate that there is no structural similarity in the vicinity of the zinc-finger motif between the XPA protein and other zinc-finger containing proteins. We conclude that the XPA protein contains a new type of zinc-finger motif.

Identification of a damaged-DNA binding domain of the XPA protein.

The XPA (xeroderma pigmentosum group A) protein is a zinc metalloprotein consisting of 273 amino acids which binds preferentially to UV- or chemical carcinogen-damaged DNA, suggesting that it is involved in the recognition of several types of DNA damage during nucleotide excision repair processes. Here we identify a DNA binding domain of the XPA protein. The region of the XPA protein responsible for preferential binding to DNA damaged by UV or cis-diammine-dichloroplatinum(II) (cisplatin) is contained within a truncated derivative of the XPA protein, MF122, consisting of 122 amino acids and containing a C4 type zinc finger motif. CD (circular dichroism) measurements of the MF122 protein showed that it has a helix-rich secondary structure, suggesting that it is a discretely folded, functional mini-domain. The MF122 protein should be useful for structural investigation of the XPA protein and of its interaction with damaged DNA.

Structure of the Oct-3 POU-homeodomain in solution, as determined by triple resonance heteronuclear multidimensional NMR spectroscopy.

The POU-homeodomain (POUH) forms the bipartite DNA-binding POU domain in association with the POU-specific domain. The 1H, 15N, and 13C magnetic resonances of the 67-amino acid long POUH of mouse Oct-3 have almost completely been assigned, mainly through the combined use of three-dimensional triple resonance NMR methods. Based on the distance and dihedral angle constraints derived from the NMR data, the solution structure of the POUH domain has been calculated by the ab initio simulated annealing method. The average RMS deviation for all backbone heavy atoms of the 20 best calculated structures for residues 9-53 of the total 67 amino acid residues is 0.44 A. The POUH domain consists of three alpha-helices (helix-I, 10-20; helix-II, 28-38; and helix-III, 42-53), and helices-II and -III form a helix-turn-helix motif. In comparison with other classical homeodomains, the folding of the three helices is quite similar. However, the length of helix-III is fairly short. In the complex of the Oct-1 POU domain with an octamer site (Klemm JD, et al., 1994, Cell 77:21-32), the corresponding region is involved in helix-III. The structural difference between these two cases will be discussed.

The XPA protein is a zinc metalloprotein with an ability to recognize various kinds of DNA damage.

The XPA (xeroderma pigmentosum group A) gene encodes a protein of 273 amino acids with a zinc finger motif. The human XPA cDNA was placed in an Escherichia coli expression vector for the synthesis of the recombinant XPA protein. The molecular weight of the wild-type protein was about 40 kDa in SDS-PAGE. Microinjection of the wild-type protein specifically restored the defect of UV-induced unscheduled DNA synthesis in XP-A cells. Thus, the bacterially expressed XPA protein retains biochemical properties identical to those of natural sources. The wild-type protein binds preferentially to UV-, cis-diamminedichloroplatinum(II) (cisplatin)- or osmium tetroxide (OsO4)-damaged DNA as assayed by retention on nitrocellulose filters. In addition, the data from atomic absorption and UV-CD spectra revealed that the wild-type protein is a zinc metalloprotein with secondary structure. Furthermore, the mutant protein, of which the cysteine-103 residue in the zinc finger motif was replaced with serine, has a vastly different protein conformation resulting in a loss of XP-A correcting and DNA-binding activities. These findings indicate that the XPA protein is a zinc-binding protein with affinity for various DNA damages, and a cysteine residue in the C4-type zinc finger motif is indispensable for normal protein conformation.

Secondary structure of the oct-3 POU homeodomain as determined by 1H-15N NMR spectroscopy.

Most of the 1H and 15N magnetic resonances of the 66 amino acid long POU homeodomain of mouse Oct-3 have been assigned by the combined use of the two-dimensional homonuclear, and two- and three-dimensional heteronuclear NMR methods. The sequential NOE connectivities and amide proton exchange measurements indicate the presence of three helical regions within the domain. The positions of the three helices correspond well to those of other homeodomains, the three-dimensional structures of which have already been determined. The present NMR study provides the first experimental evidence for the existence of a helix-turn-helix motif in the oct-3 POU homeodomain.

Infrared studies of octopus rhodopsin. Existence of a long-lived intermediate and the states of the carboxylic group of Asp-81 in rhodopsin and its photoproducts.

The infrared absorption spectra of octopus rhodopsin and its photoproducts have been observed at 282K and 210K under irradiation of blue and orange light in a neutral condition. The acid metarhodopsin-minus-rhodopsin and lumirhodopsin-minus-rhodopsin difference spectra have been obtained. A new intermediate (called transient acid metarhodopsin) with a lifetime of about 5 s has been found to exist prior to acid metarhodopsin. The present results, together with the data obtained previously, give information on the state of the carboxylic group in the side chain of Asp-81, which is the only acidic amino-acid residue in the part of opsin buried inside the membrane. This carboxylic group is protonated throughout the transformation series, but its state changes on going from transient acid metarhodopsin to acid metarhodopsin. It is probable that these two photoproducts are different from each other only in the opsin moiety.