Institute of Genetics, National Yang-Ming University, Shih-Pai, Taipei, Taiwan
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Abstract |
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Keywords: -sarcin/cassette player/domain swapping/ribonuclease T1/stranded specificity
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Introduction |
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In this study, an engineering mutant of RNase T1, denoted RNase T, is put forward. The proposed RNase T
is suggested from the concept of combining structural elements belonging to different proteins in order to generate proteins with new properties (Bennett et al., 1995
; Nixon et al., 1997
; Beguin, 1999
). Successful examples of such swapping of domains including the triggering of a chemotaxis transduction (Cochran and Kim, 1996
), the creation of a bifunctional protein that carries ß-lactamase activity in a maltodextrin-binding protein (Betton et al., 1997
), the making of a new serine protease by sub-domain shuffling (Hopfner et al., 1998
) and the acquisition of double-stranded DNA binding in cold shock protein (Wang et al., 2000
). These results have proved the feasibility of the concept. Accordingly, RNase T
, which is made up of the structure of RNase T1 except that part of its loop L3 domain has been swapped for a corresponding domain from
-sarcin, was designed to have an add-on double-stranded specificity. The ability of RNase T
to hydrolyze a single-stranded RNA and double-stranded RNA was examined in this study. The data presented not only suggest that a new ribonuclease can be created by domain swapping but also imply that RNase T1 is a nature enzyme designed to be engineered.
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Materials and methods |
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The nucleotide sequence of RNase T1 was amplified by polymerase chain reaction (PCR) from genomic DNA extracted from Aspergillus oryzae using the 5' primer 111, 5'-caaacctcgag-3'and the 3' primer 112, 5'gagagagtcccaa-3'. These primers contained XhoI and SalI restriction sites flanking their 5' and 3' termini, respectively. The PCR product was sub-cloned into the polylinker (XhoI and SalI digested) of pBluescript KS (pKS/T1) (Stratagene). After verification of the sequence, the fragment was re-cloned into pGEM (T) at the XhoI or SalI sites to make the pGEM (T)/T1 plasmid.
The chimera protein RNase T was designed to carry the structure of RNase T1 with an insertion domain derived from
-sarcin. Using a PCR-mediated strategy, two primers, the C- and N-primers were used for amplification with pGEM(T)/T1 as the template. The C-primer, 5'-AAGTTTGATTCGAAGAAGCCCAAGGAACCTggtgcgcaccgtctcctcttc-3', carries the 5' end of a sequence that encodes for the loop L3 of
-sarcin (30 bases in upper case) and the partial coding sequence of 3' end of the ß3 strand of RNase T1 (21 bases in lower case). Similarly, the sequence of the N-primer, 5'-TTCCTTGGGCTTCGAATCAAACTTgtaaacatctccggagctcagaatcg-3, carries the corresponding inserted site with the partial sequence of the loop L3 from
-sarcin (27 bases in upper case) plus 26 bases (in lower case) that encode for the 3' end of ß2 strand of RNase T1. Both primers contain a BstBI restriction site (underlined) for future self-ligation. The pGEM(T)/T1 plasmid was used as the template for the PCR amplification. The amplification was executed using the following cycling profile: denaturation (95°C for 1 min); annealing (58°C for 1 min); and extension (72°C for 3.5 min). After 25 cycles, PCR amplification yielded a linear DNA fragment of approximately 3.5 kb. The fragment was digested by DpnI restriction enzyme to eliminate endogenous pGEM(T)/T1 plasmid and further digested with BstBI restriction enzyme to generate sticky ends for the PCR fragments. Consequently, the PCR fragment was ligated to make a pGEM/T1-
plasmid that carried the coding gene for a chimeric protein, denoted RNase T
. The encoded RNase T
protein is composed of the structure of RNase T1 with its loop L3 being replaced by 10 amino acids. After the DNA sequence of the chimera gene had been confirmed, the gene was then sub-cloned into a cytosol expression pET28a vector to make a pET28a/T
plasmid.
The recombinant plasmid pET28a/T was propagated in Escherichia coli strain JM109. Cells were grown at 37°C in the presence of kanamycin (50 µg/ml) in LB broth. The expression of recombinant his-tagged RNase T
was induced by adding IPTG (250 mg/ml) when an A600 value for the culture of 0.3 was reached. The IPTG-induced cells were harvested by centrifugation. His-tagged RNase T
proteins were purified by the standard procedures of Ni chromatography. Subsequently, the his-tagged RNase T
was treated with thrombin that cleaved the carboxylic side of arginine residue of his peptide to release a recombinant RNase T
protein.
Molecular modeling
The tertiary structure of RNase T was simulated on the basis of the crystal structure of RNase T1 (Brookhaven PDB entry 1RNT) (Shirley et al., 1989
; Matinez-Oyanedel et al., 1991; Pace et al., 1991
). Using energy minimization and different conditions of constraints, the structure of RNase T
was generated. The secondary structure and the topological folding of RNase T
were almost the same as those of RNase T1. All calculations were performed on an SGI Origin 2000 computer using the program DISCOVER as implemented in the package InsightII (Molecular Simulation, San Diego, CA).
Determination of ribonuclease activity
The general ribonuclease activity measurements were carried out by RNA-impregnated SDSPAGE (Hwu et al., 2000). Purified recombinant protein was separated electrophoretically using RNA-impregnated SDSpolyacrylamide gel that contained 2.5 mg/ml of large fragments of ribosomal RNA. The presence of RNA fragments in the gel did not interfere with the resolution of protein separation. After electrophoresis, the gel was renatured by incubation in buffer containing 10 mM TrisHCl, pH 7.4 and 25% 2-propanol (to extract SDS), and this was followed by four washes with incubation buffer without 2-propanol. The gel was then incubated for 30 min at 35°C in the same buffer with vigorous shaking. The ribonuclease activity was detected by staining the gel with 2% Toluidine Blue O in water. A negative staining effect (white in color) against a blue background (the stained RNA) represented the ribonucleolytic action of protein.
The determination of the base specificity was carried out with the same RNA-impregnated SDS-containing polyacrylamide gel system, except that the gel contained monoribonucleotide polymer (poly U, poly A, poly G or poly C) (purchased from Sigma Chemical, St. Louis, MO) instead of RNA fragments.
The kinetic parameters for the transesterification of GpU dinucleoside phosphates (Sigma Chemical) were determined from initial velocities by measuring the increase in absorbance at 280 nm (Steyaert et al., 1991). The concentration of GpU dinucleoside phosphate was varied between 10 and 30 µM. Reactions were started by adding different amounts of RNase T1 or RNase T
and carried out at 35°C in buffer containing 50 mM imidazole, 50 mM NaCl and 2.5 mM EDTA at pH 6.0.
Synthesis of 34-mer oligo RNA
34-mer oligo RNA that mimics the structure of the sarcin domain from 28S rRNA was synthesized using phage T7 polymerase (Promega Biotech) and synthetic DNA oligomers as templates (England et al., 1980; Szewczak et al., 1995). The oligomers used were the sequence of T1 promoter and a sequence of 3'-ATTATGCTGAGTGATATCCCTTAGGACGAGTCATGCTCTCCTTGGCGTCCA-5' that carried a complementary T7 promoter (underlined) and the nucleotide of the sarcin domain. They were annealed at 90°C for 3 min followed by cooling on ice. The transcription reaction was carried out at 37°C for 1 h in a solution containing: 40 mM TrisHCl, pH 8.0, 9 mM MgCl2, 1 mM spermidine, 5 mM dithiothreitol, 1.5 mM each of the four nucleoside triphosphates (Pharmacia P-L Biochemical), 50 nM of the DNA template and 3 units/µl of T7 RNA polymerase. At the end of transcription, the reaction mixture containing the 34-mer oligo RNA was heated at 90°C for 2 min in buffer and renatured by gradual overnight cooling to 4°C to form a secondary structure that imitated the structure of the sarcin domain (the stem and loop structure). The 34-mer RNA was 3' end-labeled with cytidine 3',5'-[
-32P]bisphosphate according to the published procedure (Huber and Wool, 1986
).
Digestion of 34-mer RNA and 5S rRNA
The digestion of 3' end-labeled [32P]-34-mer RNA was carried out in a buffer containing 50 mM TrisHCl, pH 7.6, 50 mM KCl and 5 mM MgCl2 at 30°C for various times and using various amounts of RNase T. The cleaved oligo RNA fragments were detected with a phosphoimager. 5S rRNA from E.coli was purchased commercially and end-labeled with 32P on the 3' end accordingly. The digestion of [32P]-5S rRNA was carried out by the same procedure.
Effects of 2'-GMP on the ribonuclease activity
The nucleotide 2'-GMP is known to cognate the active site within the four-stranded pleated ß-sheet (Arni et al., 1988). Such cognition causes an inhibition of the ribonuclease activity of RNase T1. Purified recombinant enzyme was incubated with 18S rRNA (obtained from 40S subunits of rabbit reticulocyte ribosomes) in the absence or presence of different amounts of 2'-GMP in buffer containing 50 mM TrisHCl, pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA and 4% glycerol for 15 min at 30°C. The ribonuclease activity of RNase T1 or RNase T
under the influence of the 2'-GMP was analyzed using 2% agarose gel electrophoresis in TBE with ethidium bromide staining.
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Results |
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Based on similar structures of RNase T1 and -sarcin (Figure 1A
), the ribonuclease RNase T
was created by 3D domain swapping (Bennett et al., 1995
). The RNase T
carries the structure of RNase T1 except for the partial peripheral loop L3 of the RNase T1 being replaced by a corresponding loop L3 domain of
-sarcin. This was done by replacing the tetrapeptide containing SGGS (positions 6972) of RNase T1 with a sequence, KFDSKKPKEN, derived from
-sarcin (Figure 1B
). The site of replacement is within a highly conserved region (boxed in Figure 1B
) where the end residues are either glycine or proline (boxed in Figure 1B
). This makes the inserted sequence flexible enough to minimize the changes to the overall structure. The essential structure of the pleated ß-sheet is therefore maintained.
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With good results for the structure of RNase T, the gene coding for RNase T
was cloned by genetic manipulation. The amino acid sequence of cloned RNase T
was confirmed by DNA sequencing. The protein was expressed, purified, released by treatment with thrombin and then analyzed by SDS-PAGE. The recombinant RNase T
protein appears as a single band (Figure 1D
) which moves slightly slower (up-shifted) than RNase T1 in an SDS-containing polyacrylamide gel. The observed result is surprising because the difference in electrophoretic mobility does not match the predicted molecular mass of RNase T
and the difference is greater than the molecular mass of the inserted amino acid residues. Interestingly, such a slower electrophoretic mobility has also been observed in double RNase T1 mutants (Gln25
Lys; Glu58
Ala) which have a low ribonuclease activity (Shirley et al., 1989
). Thus, possibilities of changes to the structure that may affect the function of RNase T
were of deep concern. To allay this concern, structural changes in RNase T
, particularly changes to the cohesive pleated ß-sheet structure which carries the catalytic center of RNase T1, were analyzed using circular dichroism (CD). The CD measurements showed that RNase T
has similar CD spectra to RNase T1 (Figure 2
), indicating that RNase T
seems to have adopted the same conformation as RNase T1 in solution.
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The hydrolysis of 18S rRNA described above implies that RNase T is an effective ribonuclease of naked RNA. Therefore, the base specificity of RNase T
was examined next. Using RNA-impregnated SDSPAGE, it was found that RNase T
, in addition to hydrolyzing naked RNA (Figure 4A
), also effectively hydrolyzed polyguanosine (Figure 4B
), but not polycytidine (Figure 4C
), polyuridine (data not shown) or polyadenosine (data not shown). These results indicate that RNase T
has preserved the guanyl specificity of RNase T1.
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As suggested from the initial notion of this study, the question of whether RNase T with its swapped domain had enhanced ribonuclease activity towards double-stranded RNA was therefore examined. To assess this experiment, a 3' end-labeled synthetic 34-mer oligo RNA (Figure 5A
) was used as the substrate for RNase T
. The 34-mer oligo RNA represents the stem/loop sarcin domain of large subunit ribosomal RNA, which is the best known substrate for
-sarcin (Endo et al., 1988
; Correll et al., 1999
; Perez-Canadillas et al., 2000
). The result on the hydrolysis of 34-mer oligo RNA by RNase T
was as predicted. It was found that RNase T
initially cleaved single-stranded guanine bases at the 5' end (G2 and G3) to produce a 32-mer and a 31-mer, respectively (Figure 5B
). At increasing concentration, RNase T
cleaved at single-stranded G21 of the tetra loop in a manner similar to that for
-sarcin (Endo et al, 1988
; Cheung et al., 1996
). Moreover, RNase T
also hydrolyzed the double-stranded guanine base G23 in addition to G24, a non-WatsonCrick A17:G24 pairing (Correll et al., 1999
), releasing an 11-mer and a 10-mer, respectively (Figure 5B
). In a comparative experiment, RNase T1 showed limited ribonuclease activity at the single-stranded guanine bases, acting at G2 and G3 near the 5' end and at G21 within the tetra loop (Figure 5B
).
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Discussion |
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The success of engineered RNase T is significant in many aspects. Practically, the engineered RNase T
is a potential tool for footprinting the structure of ribonucleicprotein complexes (RNP). The use of
-sarcin with its lack of strand preference and purine specificity has helped to elucidate in detail the structures of the 5S rRNP (Huber and Wool, 1986
), the 7S rRNP (Sands and Bogenhagen, 1991
) and the SRP (signal recognition particle) (Stub et al., 1991). Ironically, the properties of the engineered RNase T
, with it lack of strand preference and guanyl specificity, make RNase T
a better tool than
-sarcin for footprinting the RNP complex. An experiment using of RNase T
to dissect an RNP complex is in progress.
In essence, the most significant aspect of this study, besides proving the feasibility of making a new ribonuclease by domain swapping, is the discovery that the part of the loop L3 in the RNase T1 can be exchanged without jeopardizing its ribonuclease activity. In numerous studies of RNase T1, the activities of single, double or triple mutations on RNase T1 have been reported (Grunert et al., 1991; Steyaert and Wyns, 1993
; Loverix et al., 1997
, 1998
; Steyaert, 1997
; Hubner et al., 1999
); however, such an exchange of a partial domain has never previously been carried out. Our example of an engineered RNase T
suggests that RNase T1 is an enzyme capable of further engineering. The potential use of this work can be predicted where RNase T1 becomes a cassette player and the loop L3 is a site for where new molecular cassettes are inserted. This will allow the creation of a series of novel enzymes with new activities. Any functional module could be considered as a cassette to slot into the position of the loop L3 of RNase T1. Then that RNase T1 would be expected to play the activity of an inserted module along with the original ribonuclease activity. Ultimately, in the future it should be possible to generate a wide range of specific ribonucleases using this concept.
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Notes |
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Acknowledgments |
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References |
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Received May 28, 2002; revised September 26, 2002; accepted October 10, 2002.