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Expression, purification and structural analysis of the Pyrococcus abyssi RNA binding protein PAB1135
© Oliveira et al; licensee BioMed Central Ltd. 2010
- Received: 20 November 2009
- Accepted: 9 April 2010
- Published: 9 April 2010
The gene coding for the uncharacterized protein PAB1135 in the archaeon Pyrococcus abyssi is in the same operon as the ribonuclease P (RNase P) subunit Rpp30.
Here we report the expression, purification and structural analysis of PAB1135. We analyzed the interaction of PAB1135 with RNA and show that it binds efficiently double-stranded RNAs in a non-sequence specific manner. We also performed molecular modeling of the PAB1135 structure using the crystal structure of the protein Af2318 from Archaeoglobus fulgidus (2OGK) as the template.
Comparison of this model has lead to the identification of a region in PAB1135 that could be involved in recognizing double-stranded RNA.
- Uncharacterized Protein
- Stability Secondary Structure
- Circular Dichroism Analysis
- Electrophoresis Mobility Shift Assay
- PAB1135 Gene
Despite the recent progress in various genome analysis projects, about a quarter of the archaeal genomes encode functionally uncharacterized proteins, which are almost all only common to other archaeal species [1–4]. Pyrococcus abyssi PAB1135 protein function has not yet been characterized, and it is classified in the family domain of unknown function 54 (DUF54) and in the uncharacterized protein family 0201 (UPF0201). This group's members have been annotated as conserved hypothetical proteins in 46 archaeal species. Some of these proteins were annotated as possible exosome subunits (TK1451 - GI: 57641386, Thermococcus kodakarensis; MK0388 - GI: 20093826, Methanopyrus kandleri; Msp1244 - GI: 84490032, Methanosphaera stadtmanae) [4–6], but analysis of completely sequenced Pyrococcus abyssi genome revealed the presence ofPAB1135 gene in the same operon as Pa1136, the ribonuclease P (RNase P) subunit Rpp30 .
RNase P is an endoribonuclease responsible for maturation of tRNAs in all domains of life. RNase P is a ribonucleoprotein (RNP) complex, formed by one RNA molecule and a variable number of protein subunits, depending on the organism. Bacterial RNase P contains one protein, whereas the archaeal relative contains at least four proteins, and in humans, it contains at least 10 protein subunits [8, 9]. Pyrococcus horikoshii RNase P has been shown to be formed by one catalytic RNA and the proteins Ph1481, Ph1601, Ph1771, and Ph1877, which show homology to the human RNase P subunits hPop5, Rpp21, Rpp29, and Rpp30, respectively . The structures of these P. horikoshii proteins have been solved and a possible arrangement of the protein complex has been proposed .
Although the function of Pyrococcus abyssi PAB1135 has not been characterized, nor its association with RNase P complex, the genome location of the gene suggests that PAB1135 is involved in RNA metabolism. Here we show that PAB1135 binds RNA in vitro, showing higher affinity for double-stranded RNAs. In addition, structural analysis of PAB1135 by molecular modeling indicates a possible region for protein-RNA interaction.
Cloning of PAB1135 sequence
The Escherichia coli strains used in this study were DH5α and BL21-CodonPlus (DE3)-RIL (Stratagene). Plasmid DNA was extracted using Qiagen plasmid purification systems. Restriction enzymes and other DNA-modifying enzymes were used as recommended by the manufacturer (New England Biolabs). PAB1135 coding sequence was PCR-amplified from P. abyssi GE5 genomic DNA Genomic DNA (kindly provided by Dr. Patrick Forterre from Institut de Génétique et Microbiologie, Université Paris Sud, France) using primers PAB1135for (5'-GTTAGGGGGGATCC ATG GCAG-3') and PAB1135rev (5'-CGGCCTCGA GTCAAT CCTCCC-3'). The restriction sites used are underlined in the primers' sequences, and the start and stop codons are in bold. A DNA fragment of 462 bp was obtained from the PCR reaction and inserted into vector pET28a (Novagen), digested with BamH I-Xho I. A 21 kDa tagged protein His-PAB1135 was produced from this plasmid
Expression and purification of the recombinant protein
The pET28a-PAB1135 was transformed into the E. coli BL21-CodonPlus (DE3)-RIL strain. The transformed cells were grown at 37°C in 2xTY medium supplemented with 20 mg/L kanamycin and 17 mg/L chloramphenicol. The expression of His-PAB1135 was induced for two hours with 0.5 mM IPTG. Cells were harvested by centrifugation, suspended in buffer A (30 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole) and lysed in a French press. The lysate was heated at 85°C for 30 min and cooled on ice for 15 min. After centrifugation at 20,000 × g for 30 min, the supernatant was fractionated by affinity chromatography in Ni-NTA-agarose (Qiagen). The purified fractions were analyzed by SDS-PAGE.
Thrombin and Trypsin digestion and analysis
To remove the His-tag from PAB1135, tagged protein was incubated with thrombin for 8 h at room temperature, as recommended by the manufacturer (GE Healthcare). Trypsin treatment was performed by incubating His-PAB1135 with 0.1% trypsin solution (0.1 mg/ml) for 2 hours at room temperature, followed by the addition of 1 mM PMSF. His-PAB1135 and its tryptic cleavage products were subjected to SDS-PAGE and transferred to PVDF membranes (BioRad), which were incubated with an anti-poly-histidine antibody (GE Healthcare). The immunoblots were developed using the ECL system (GE Healthcare).
Gel filtration and circular dichroism
Gel filtration assays of trypsin-treated PAB1135 were carried out in a superdex 75 XK 16/60 column (GE Healthcare) in the presence of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM EDTA. Apparent molecular masses were assessed based on the retention time of the molecular mass markers (low molecular mass gel filtration calibration kits, GE Healthcare: bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; chymotrypsinogen, 25 kDa; ribonuclease A, 13.7 kDa).
For the circular dichroism analysis, PAB1135 was dialyzed against buffer B (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 5 mM MgCl2, 1% glycerol, 0.02% tween 20, 1 mM EDTA) and concentrated to 2 mg/ml using Amicom ultra (Millipore). The circular dichroism experiments were conducted on a JASCO 810 spectropolarimeter using a 1 mm path length cuvette. The K2d program  was used for the estimation of the percentages of protein secondary structure from circular dichroism data. Several CD curves were generated using the algorithm proposed by  to obtain best fit with the experimental data. The CD deconvolution and generation of CD curves were performed using the K2d program at http://www.embl.de/~andrade/k2d/.
RNA binding assays
RNA binding assays were carried out with 1 pmol 32P 5'-labeled oligoribonucleotides. The oligos used were: U8C5A8 (5'UUUUUUUUCCCCCAAAAAAAA3'), C8U5G8 (5'CCCCCCCCUUUUUGGGGGGGG3'), and UUA/C (5'UUAUUAUUCAUUCAUUAUUCA3'). The assays were performed as described previously [14, 15], in Tris-HCl pH 8.0, 20 mM KCl, 5 mM MnCl2, 1 mM DTT, 100 ug/ml BSA, 0.8 U Rnasin. Different amounts of trypsin-treated PAB1135 were incubated with the RNA oligos in 20 μl at 37°C for 30 minutes. The samples were resolved on 8% native polyacrylamide gels and visualized on a Phosphorimager (MolecularDynamics).
Molecular modeling and structural analysis
MODELLER [, version 9v1] was used to produce a homology molecular model of PAB1135. The structure of the conserved hypothetical protein Af2318 from Archaeoglobus fulgidus (PDB code 2OGK, ) was used as a template. This protein shares 40% identity with the PAB1135 sequence, being the top hit in a search through the PDB  using BLAST . The parameters used during the modeling exercise were the default of the programs. The alignment used in MODELLER was produced with CLUSTALX . The alignments of the N- and C-terminal regions were cut at the corresponding ends of the crystallographic model.
The homology models were validated with the VERIFY-3D  and PROCHECK  softwares. The analysis was made through visualization of the superimposed structures using PYMOL  and various alignments produced with CLUSTALX. COOT  was used for the superposition  of the atomic coordinates of the models and PDB files: 2OGK, 1JJ2, and 1MJI. DALI  was also used for analysis of correlated structures.
Purification of PAB1135
Interestingly, results from gel filtration assays suggest that PAB1135 forms a homodimer in solution, with an apparent molecular weight of about 30 kDa (Figure 2B), whereas a PAB1135 monomer runs as an approximately 20 kDa protein on SDS-PAGE. This is very similar to the calculated 35.8 kDa molecular weight of a homodimer. In the conditions tested, PAB1135 appears monodisperse in solution since 100% of the mass was accounted for by a single peak at 2.7 nm. The temperature variation between 25°C and 50°C did not affect the results.
We show that PAB1135 is highly conserved and its structure can be inferred by molecular modeling based on the crystal structures of archaeal UPF0201 proteins. Furthermore, as shown here, PAB1135 binds RNA in vitro, with higher affinity for structured RNAs, in accordance with the model's suggestion for the presence of an RNA binding scaffold in the protein. It is possible that PAB1135 binds structured RNAs in vivo, such as RNase P RNA and tRNAs.
This work was supported by a FAPESP grant (07/57096-9 to C.C.O.). J.S.L. was recipient of a FAPESP fellowship.
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