WO2007010740A1

WO2007010740A1 - Novel endoribonuclease

Info

Publication number: WO2007010740A1
Application number: PCT/JP2006/313270
Authority: WO
Inventors: Masamitsu Shimada; Masanori Takayama; Kiyozo Asada; Ikunoshin Kato
Original assignee: Takara Bio Inc.
Priority date: 2005-07-21
Filing date: 2006-07-04
Publication date: 2007-01-25
Also published as: JPWO2007010740A1

Abstract

A polypeptide having a novel endoribonuclease activity; a nucleic acid encoding the polypeptide; recombinant DNA having the nucleic acid therein; a transformant transformed with the recombinant DNA; a process for producing the polypeptide comprising the steps of cultivating the transformant and collecting the polypeptide from the culture; a process for producing a digest of single-stranded RNA comprising the step of reacting the polypeptide with the single-stranded RNA; and a method for the digestion of single-stranded RNA.

Description

Specification

Novel endoribonuclease

Technical field

[0001] The present invention relates to a novel sequence-specific endoribonuclease useful in the field of genetic engineering.

Background art

[0002] V, some prokaryotic plasmids have been reported to have post-segregation killing (PSK) functions that kill the host from which the plasmid was dropped in order to maintain the plasmid in the host. These plasmids contain the toxin antitoxin gene. Anti-toxin binds with the toxin in the cell and inactivates the toxin Anti-toxin is prone to degradation by protease, and when it is degraded by protease, stable toxin is activated (non- Patent Document 1). Such a toxin-antitoxin gene is also present in most prokaryotic chromosomes, supports various stresses, and plays a role in programmed cell death. Although the functions of these toxins have not yet been clarified, it has been suggested that CcdB and ParE may regulate replication using DNA gyrase as a target, while RelE and Doc may regulate transcription. (Non-Patent Documents 1 and 2).

[0003] In E. coli, there are at least five toxins of RelE, ChpAK (MazF), ChpBK, YoeB, and YafQ (Non-patent Document 2). Christensen et al. Reported that RelE is an endoribonuclease that cleaves mRNA by recognizing a specific codon of 3 bases depending on ribosome (Non-patent Documents 3 and 4). Christensen et al. Also reported that ChpA :, ChpBK and YoeB are also endoribonucleases that cleave mRNA in a ribosome- and codon-dependent manner (Non-Patent Documents 5 and 6).

[0004] On the other hand, Inoue et al. Proved that MazF (ChpAK) is an endoribonuclease that recognizes a specific base of ACA and cleaves mRNA in a ribosome-independent manner (Non-Patent Document 7, 8). Munoz-Gomez et al. Reported that the specificity of mazF for RNA cleavage is NAC (Non-patent Document 9). Furthermore, Inoue et al. Described Pe present in plasmid R100. It has been proved that mK is an endoribonuclease that recognizes a specific base of UAH (H is C, A or U) and cleaves mRNA (Patent Document 1, Non-Patent Document 10). As described above, it has been suggested that RelE and PemK family toxins may be endoribonucleases that cleave mRNA in a base-specific manner. In particular, the PemK family of toxins may be endoribonucleases that recognize specific bases and cleave mRNAs independently of ribosomes. Many PemK family toxins are present in prokaryotes, and their sequence comparison has been well studied (Non-patent Documents 1 and 11).

[0005] In addition, Anantharaman et al. Performed gene neighborhood analysis based on the genetic information of toxins and the genetic information of genomes for which genome analysis was completed, systematically classifying the toxins, and also for proteins with unknown functions. A toxin-like protein was predicted (Non-patent Document 12). Further analysis suggests that not only RelE and PemK, but also proteins with the Doc family and PIN domains may have ribonuclease activity. Bacillus subtilis has one PemK family toxin, Nostoc sp. Has four PemK family toxins, and Enterococcus faecalis has three PemK family toxins (Non-patent Document 13). Mathy et al. Found that the Bacillus subtilis PemK family toxin YdcE is an endoribonuclease and reported that it is similar to MazF and recognizes the UAC sequence (Non-patent Document 14).

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[0006] As an enzyme that cleaves nucleic acids in a sequence-specific manner, many restriction enzymes that cleave double-stranded DNA have been found and are widely used in the field of genetic engineering. An enzyme that specifically cleaves single-stranded RNA has been found in ribonuclease T1, which specifically cleaves G bases and is used in genetic engineering (Non-patent Document 15). In the field of genetic engineering, there are still few enzymes that recognize and specifically cleave a plurality of bases, and development of such endoribonucleases is desired. If an endoribonuclease that specifically recognizes and cleaves a 3 base sequence such as MazF or more is discovered, it will be a useful enzyme in the field of genetic engineering.

[0007] Patent Document 1: Pamphlet of International Publication No. 2004Z113498

Non-Patent Document 1: Journal 'Ob' Battereriol. (J. Bacteriol.), 182, p5 61-572 (2000)

Non-Patent Document 2: Science, No. 301, pl496-1499 (2003) Non-Patent Document 3: Molecular Microbiol., No. 48, pl389-1400 (2003)

Non-Patent Document 4: Cell, 122, 131-140 (2003)

Non-Patent Document 5: Journal 'Ob' Molequila 'Biology. Mol. Biol.), No. 332, p809-819 (2003)

Non-Patent Document 6: Molecular One's Microbiology, No. 51, pl705-1717 (2004) Non-Patent Document 7: Molecular Cell, No. 12, p913-920, 200 3)

Non-Patent Document 8: Journal 'Ob' Biological 'Chemistry (J. Biol. Chem.), No. 280, p3143-3150 (2005)

Non-Patent Document 9: FEBS Letters, No. 567, p316-320 (2004)

Non-Patent Document 10: Journal 'Ob' Biological 'Chemistry, Vol. 279, p20678 -20684 (2004)

Non-Patent Document 11: Journal · Bob · Molequila 'Microbiology' and Biotechnology (J. Mol. Microbiol. Biotechnol.), No. 1, p295-302 (1999)

Non-patent document 12: Genome Biology, IV, R81 (2003) Non-patent document 13: Nucleic Acids Research, Vol. 33, p966-976 (2005)

Non-Patent Document 14: Molequila 'Microbiology, No. 56, pi 139— 1148 (2005)

Non-Patent Document 15: Method in Enzymology, No. 3 41, p28-41 (2001)

Disclosure of the invention

Problems to be solved by the invention [0008] An object of the present invention is to provide a novel sequence-specific endoribonuclease in view of the above prior art, and the specificity of the novel sequence-specific endoribonuclease cleavage sequence. Is to provide and use for genetic engineering.

Means for solving the problem

[0009] The present inventors screened a sequence-specific endoribonuclease, and the polypeptide encoded by each gene of Nostoc s p. A113211, Bacillus subtilis YdcE and Enterococcus faecalis EFO 850 was a novel sequence-specific. Was found to be a typical endoribonuclease. Furthermore, the specificity of the cleavage sequence of the enzyme was identified and the present invention was completed.

[0010] That is, the present invention provides:

[1] Amino acid sequence described in SEQ ID NO: 1, 2, or 3 in the sequence listing, or an amino acid sequence having at least one deletion, addition, insertion or substitution of one or more amino acid residues in the sequence And a polypeptide having sequence-specific endoribonuclease activity,

[2] a nucleic acid encoding the polypeptide of [1],

[3] The nucleic acid according to [2], having the base sequence described in SEQ ID NO: 4, 5 or 6 in the sequence listing,

[4] A nucleic acid encoding a polypeptide capable of being or hybridized under stringent conditions to the nucleic acid of [2] or [3] and having sequence-specific endoribonuclease activity,

[5] [2] to [4] V, shear force Recombinant DNA comprising the nucleic acid according to item 1,

[6] A transformant transformed with the recombinant DNA of [5],

[7] The polypeptide according to [1], comprising the step of culturing the transformant according to [6] and the step of collecting a polypeptide having a sequence-specific RNA cleavage activity from the culture. Manufacturing method,

[8] A method for producing a single-stranded RNA degradation product, comprising the step of allowing the polypeptide of [1] to act on single-stranded RNA; and

[9] A method for degrading single-stranded RNA, comprising the step of allowing the polypeptide of [1] to act on single-stranded RNA, About.

The invention's effect

[0011] According to the present invention, it is possible to find a novel sequence-specific endoribonuclease, identify the specificity of the cleavage sequence of the novel sequence-specific endoribonuclease, and provide use for genetic engineering.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] 1. Polypeptide of the present invention

The polypeptide of the present invention is an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1, 2 or 3, or an amino acid sequence having at least one deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence. It is characterized by its sequence-specific and sequence-specific endoribonuclease activity.

[0013] The activity of the polypeptide of the present invention is a single-stranded RNA-specific endoribonuclease activity, and the activity of ribonucleotides in a single-stranded nucleic acid containing ribonucleotides as a constituent base. 'The phosphodiester bond on the side can be hydrolyzed. The nucleic acid hydrolyzed by the activity has a 3 ′ end having a hydroxyl group and a 5 ′ end having a phosphate group, a 3 ′ end having a phosphate group and a 5 ′ end having a hydroxyl group, or a 2 ′ or 3 ′ site. This produces a 5 'end with click phosphate and hydroxyl groups.

[0014] The substrate of the polypeptide of the present invention may be a nucleic acid having at least one molecule of ribonucleotide, such as RNA, RNA containing deoxyribonucleotide, DNA containing ribonucleotide, and the like. It is not limited to these. The substrate contains a nucleotide different from that contained in a normal nucleic acid within a range not inhibiting the action of the polypeptide of the present invention, such as deoxyinosine, deoxyuridine, hydroxymethyldeoxyuridine and the like. Well, okay.

[0015] The polypeptide of the present invention specifically acts on a single-stranded nucleic acid. Double-stranded nucleic acids, such as double-stranded RNA, RNA-DNA nobled, etc. cannot be cleaved! /.

[0016] The polypeptide of the present invention is characterized by having an activity of cleaving a nucleic acid in a specific base sequence. Although the present invention is not particularly limited, for example, a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, The peptide and the polypeptide having the amino acid sequence shown in SEQ ID NO: 3 are located at the 5 ′ end of the sequence when a 5′-UACAU-3 ′ sequence is present in the single-stranded RNA molecule. It hydrolyzes the phosphodiester bond between the residue and the 3 'adjacent A residue. This activity is confirmed as an activity of hydrolyzing a phosphodiester bond between the 14th and 15th bases of the oligoribonucleotide using, for example, mazG30, which is an oligoribonucleotide having the base sequence shown in SEQ ID NO: 13, as a substrate. can do. In addition, the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 hydrolyzes the phosphodiester bond between the U residue and the A residue for the 5′-UACA-3 ′ sequence in the single-stranded RNA molecule. Can be solved. Since the endoribonuclease activity of the polypeptide of the present invention is expressed without the coexistence of ribosomes, the activity is ribosome-independent.

[0017] The single-stranded RNA-specific endoribonuclease activity of the polypeptide of the present invention can be measured, for example, using single-stranded RNA as a substrate. Specifically, a single-stranded RNA transcribed with RNA polymerase in the shape of a DNA or a chemically synthesized single-stranded RNA is allowed to act on a polypeptide whose activity is to be cleaved. It can be measured by examining whether it occurs. RNA degradation can be confirmed, for example, by electrophoresis (agarose gel, acrylamide gel, etc.). If a suitable label (for example, a radioisotope, a fluorescent substance, etc.) is attached to RNA as a substrate, it becomes easy to detect degradation products after electrophoresis.

[0018] The polypeptide of the present invention has one or more amino acid sequences described in SEQ ID NO: 1, 2 or 3 in the sequence listing as long as it exhibits endoribonuclease activity that hydrolyzes single-stranded RNA in a sequence-specific manner. It includes a polypeptide represented by an amino acid sequence in which at least one of deletion, addition, insertion or substitution of amino acid residues has been made. Examples of the polypeptide having such a mutation include a polypeptide having 50% or more homology to the polypeptide described in SEQ ID NO: 1, 2, or 3, preferably a polypeptide having 70% or more homology, Particularly preferred is a polypeptide having 90% or more homology. Polypeptides having these mutations are included in the present invention even if they recognize and cleave a sequence different from the polypeptide of the amino acid sequence described in SEQ ID NO: 1, 2 or 3.

[0019] Further, the polypeptide has a peptide region essential for its activity! /, For example, peptides for improving the efficiency of translation, peptides for facilitating purification of the polypeptide (for example, histidine tag, dartathione S transferase, maltose binding protein, etc.), chaperones, etc. Even those added with a tank are included in the polypeptide of the present invention as long as they exhibit RNA-cleaving activity specific to single-stranded RNA.

[0020] 2. Nucleic acid encoding the polypeptide of the present invention

The present invention provides a nucleic acid encoding a polypeptide having sequence-specific endoribonuclease activity. The nucleic acid is not particularly limited, but the amino acid sequence described in SEQ ID NO: 1, 2, or 3 in the sequence listing, or one or more, for example, 1-10 amino acid residues missing from the sequence. Examples thereof include those which are represented by an amino acid sequence having at least one of deletion, addition, insertion or substitution, and which encode a polypeptide having the sequence-specific endoribonuclease activity. Here, as the amino acid sequence having at least one of deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence described in SEQ ID NO: 1, 2 or 3, for example, SEQ ID NO: 1, 2 or An amino acid sequence having 50% or more homology in the polypeptide described in 3, preferably an amino acid sequence having 70% or more homology, particularly preferably an amino acid sequence having 90% or more homology.

Furthermore, the nucleic acid of the present invention includes a nucleic acid that can hybridize to the above-mentioned nucleic acid under stringent conditions and encodes a polypeptide having sequence-specific endoribonuclease activity. The above stringent conditions are as follows: 1989, Cold 'Spring' Nova 1 'Laboratory published, edited by J. Sambrook et al., Molecular ^ ~ · Cloning:' Laboratory ^ ~ · Examples include conditions described in the second edition of the manual (Molecular Cloning: A Laboratory Manual 2nd ed.). Specifically, for example, conditions of incubation with a probe at 65 ° C. for 12 to 20 hours in 6 X SSC containing 0.5% SDS, 5 X Denharz solution, and 0.01% denatured salmon sperm DNA can be mentioned. The nucleic acid hybridized to the probe can be detected after removing non-specifically bound probe by washing at 37 ° C. in 0.1 × SSC containing 0.5% SDS, for example. [0022] The nucleic acid encoding the polypeptide of the present invention can be obtained, for example, by the following means.

[0023] A gene having homology in the amino acid sequence to a toxin such as MazF or PemK that has endoribonuclease activity that recognizes a specific base sequence and cleaves mRNA is a polypeptide having sequence-specific ribonuclease activity. Candidate nucleic acids to encode. Such candidate genes can be found, for example, from the bacterial genome. Bacillus subtilis has one PemK family toxin, Nostoc sp. Has four PemK family toxins, and Enterococcus faecalis has three PemK family toxins.

[0024] Candidate genes can also be isolated from bacterial genomic strength by PCR using primers designed based on nucleotide sequence information, for example. If the entire base sequence is known, the entire sequence of the candidate gene can be synthesized using a DNA synthesizer.

[0025] Protein expression with candidate gene ability can be carried out in an appropriate host transformed with an expression vector incorporating the candidate gene, for example, E. coli. Expression of sequence-specific ribonucleases that degrade host RNA may be lethal to the host, and the expression of candidate genes must be strictly suppressed until induction. For example, it is preferable to use an expression system such as a pET system (manufactured by Novagen) using a T7 polymerase promoter or a cold shock expression control system pCold system (manufactured by Takara Bio Inc.). In order to easily purify the expression product from the candidate gene, it is advantageous to add a peptide such as the histidine tag to the expression product in order to facilitate the purification. For this purpose, an expression vector containing such a peptide coding region may be used.

[0026] The measurement of endoribonuclease activity can be carried out by the above-described method using single-stranded RNA as a substrate. The cleavage site can be identified by a primer extension using a cleaved RNA as a saddle and a primer complementary to the RNA and reverse transcriptase. Since the extension reaction stops at the cleavage site in the primer extension, the cleavage site can be identified by determining the length of the extended strand by electrophoresis. Furthermore, in order to precisely identify the nucleotide sequence specificity, an oligoribonucleosome having an arbitrary sequence is used. After chemically synthesizing the tide and allowing the expression product of the candidate gene to act, the presence or absence of cleavage may be determined by denaturing acrylamide gel electrophoresis or the like.

[0027] 3. Method for producing polypeptide of the present invention

The polypeptide of the present invention is, for example, (1) purified from a culture of a microorganism producing the polypeptide of the present invention, or (2) capable of culturing a transformant containing a nucleic acid encoding the polypeptide of the present invention. It can be produced by a method such as purification.

[0028] The microorganism that produces the polypeptide of the present invention is not particularly limited, and examples include bacteria belonging to the genera Bacillus, Nostoc and Enterococcus. For example, the polypeptide of the present invention can be obtained from Bacillus suDtilis, Nostoc sp., Enterococcus faecaiis, particularly preferably Baku subtilis 168 strain, Nostoc sp. PCC7120 strain, E. faecalis V583 strain. The culture of the microorganism should be performed under conditions suitable for the growth of the microorganism. The target polypeptide produced in the bacterial cells or the culture solution can be obtained by methods commonly used for protein purification, such as disruption of bacterial cells, fractionation by precipitation (such as ammonium sulfate salting-out), and various chromatography (ion It can be purified by exchange chromatography, affinity chromatography, hydrophobic chromatography, molecular sieve chromatography, etc.) or a combination thereof.

[0029] The polypeptide of the present invention can be obtained from the transformant transformed with the recombinant DNA containing the nucleic acid encoding the polypeptide of the present invention. The recombinant DNA is preferably provided with a suitable promoter functionally connected upstream of the nucleic acid encoding the polypeptide of the present invention. Since the polypeptide of the present invention may have a lethal action on the host, the expression system including the promoter and promoter described above strictly transcribes the nucleic acid that encodes the polypeptide of the present invention. It is preferably one that can be closely controlled. Examples of such a system include the pET system and the pCold system.

[0030] The above recombinant DNA may be introduced as it is into a host cell, and may be introduced by being inserted into an appropriate vector such as a plasmid vector, a phage vector, or a virus vector. Furthermore, the above recombinant DNA may be integrated into the host chromosome. The host to be transformed is not particularly limited, for example, E. coli, Bacillus subtilis, yeast, Examples include hosts that are commonly used in the field of recombinant DNA, such as filamentous fungi, plants, animals, plant culture cells, and animal culture cells.

[0031] The polypeptide of the present invention produced by these transformants can be purified using the purification method as described above. When the nucleic acid encoding the polypeptide of the present invention encodes a polypeptide to which a peptide for facilitating the purification of the polypeptide is added, purification becomes very easy. By using a purification method according to the added peptide, for example, using a metal chelate resin for histidine-tag and a dartathione-fixed resin for daltathione S-transferase, respectively, A pure polypeptide can be obtained by a simple operation.

[0032] 4. Degradation of single-stranded RNA using the polypeptide of the present invention

By using the polypeptide of the present invention, single-stranded RNA can be degraded to produce an RNA degradation product. Since the polypeptide of the present invention can cleave RNA in a base sequence-specific manner, the average chain length of the generated RNA degradation product correlates with the appearance frequency of the base sequence recognized by the polypeptide. That is, the present invention provides an RNA degradation product having a certain chain length distribution. Furthermore, it is possible to excise a specific region in RNA using its sequence specificity.

[0033] Furthermore, single-stranded RNA can be selectively degraded by the polypeptide of the present invention.

As one embodiment of the present invention, protein synthesis systems, for example, cell-free translation systems and mRNAs in transformants can be degraded with the polypeptide of the present invention to inhibit protein synthesis. At this time, the mRNA encoding the desired protein, which is artificially prepared so as not to contain the base sequence recognized by the polypeptide of the present invention, is allowed to exist in the above system, so that only the mRNA is degraded. The desired protein is specifically produced in the system. This embodiment is particularly useful for producing highly pure protein. The polypeptide of the present invention has the ability to cleave single-stranded RNA. It does not cleave double-stranded RNA or RNA-DNA hybrids! This property can be used to analyze the secondary structure of RNA. For example, when the polypeptide of the present invention is allowed to act on the RNA to be analyzed, if the sequence cleavable by the polypeptide of the present invention in the RNA molecule is not cleaved, the above sequence At least a part of the base pair bond with other sequences Is expected. tRNA forms a secondary structure called the cloverleaf model, and the three loop structures are single-stranded. A sequence that can be cleaved by the polypeptide of the present invention present in tRNA is cleaved by the polypeptide of the present invention only when the sequence is located in the loop region. Utilizing this fact, tRNA identification and structural analysis using the polypeptide of the present invention can be carried out.

Example

[0034] The ability to describe the present invention more specifically with reference to the following examples The present invention is not limited to the following examples.

[0035] Among the operations described in this specification, the basic operations are described in 2001, published by Cold Spring Harbor Laboratory, edited by J. Sambrook et al., Molecular Cloning: According to the method described in Laboratory Manual 3rd Edition (Molecular Cloning: A Laboratory Manual, 3rd ed.).

Example 1 Isolation of Bdc subtilis 168 strain YdcE, Nostoc sp. PCC7120 strain All 3211, E. faecalis V583 strain EF0850 isolation and expression plasmid construction

The amino acid sequence and nucleotide sequence of the polypeptide obtained from ydcE from Bacillus subtilis 168, all3211 from Nostoc sp. PCC7120, and EF0850 from Enterococcus faecalis V583 were obtained from the NCBI database. (Accession No. NP—388347 and NC—000964, NP—487251, and NC—003272, NP—814592 and NC—004668). Based on the nucleotide sequence information of ydcE, all3211, EF0850, primer ydcE—F (SEQ ID NO: 7) and primer ydcE —R (SEQ ID NO: 7) are used for ydcE so that the DNA of the region encoding the entire polypeptide can be amplified by PCR. 8), primer all3211—F (SEQ ID NO: 9) and primer all3211—R (SEQ ID NO: 10) for all3211, primer EF0850—F (SEQ ID NO: 11) and primer EF0850—R (SEQ ID NO: 12) for EF0850 Each was synthesized.

[0037] Genomic DNA was prepared from B. subtilis 168 strain and Nostoc sp. PCC7120 strain by the following method. The cells were suspended in 0.5 ml of TE buffer containing 50 U of mutanolysin (manufactured by Nacalai Testa) and 0.1 mg of lysozyme (manufactured by Sigma), incubated at 37 ° C for 2 hours, and then 10% SDS50; zl, 10mg / ml proteinase K5 1 Incubated for an additional hour. Next, phenol extraction and black mouth form extraction were performed, followed by ethanol precipitation to obtain genomic DNA. Genomic DNA was dissolved in TE buffer. E. faecalis V583 strain genomic DNA was obtained from ATCC (ATCC No. 700 802D).

PCR using Pyrobest DNA polymerase (manufactured by Takara Bio Inc.) was performed using 50 ng of B. subtilis 168 genomic DNA, primers ydcE-F and ydcE-R, and an amplified DNA fragment of 371 bp was obtained. This amplified fragment was digested with restriction enzymes Ndel and Xhol and subjected to agarose electrophoresis, and a 350 bp DNA fragment was recovered from the gel after electrophoresis. In the same manner, a 461 bp amplified fragment and a 440 bp restriction enzyme digested DNA fragment were obtained using Nostoc sp. PCC7120 strain genomic DNA, primers all321 1 -F and all3211-R. In addition, a 398 bp amplified fragment and a 377 bp restriction enzyme digested DNA fragment were obtained using E. faecalis V583 strain genomic DNA and primers EF08 50-F and EF0850-R. After digestion with restriction enzymes Ndel and Xhol! And transformation of Escherichia coli JM109 with the recombinant plasmid obtained by connecting the above 350bp, 440bp or 377bp DNA fragment to pET21a vector (Novagen). did. Plasmids were prepared from the thus obtained transformant colonies, their nucleotide sequences were confirmed, and these were designated expression vectors pET-ydcE, pET-all3211 and pET-EF0850, respectively.

The base sequence encoding the YdcE polypeptide derived from the B. subtilis 168 strain inserted into the expression vector pET-ydcE is shown in SEQ ID NO: 5, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 2, respectively. The nucleotide sequence encoding the A113211 polypeptide derived from Nostoc sp. PC C7120 strain inserted into the expression vector pET-all3211 is shown in SEQ ID NO: 4, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, respectively. The base sequence encoding the EF0850 polypeptide derived from the E. faecalis V583 strain inserted into the expression vector pET-EF0850 is shown in SEQ ID NO: 6, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 3, respectively. According to the expression vectors pET—ydcE, pET—all3211 and pET—EF0850, the polypeptide having the amino acid sequence of SEQ ID NO: 1, 2 or 3 also has an 8 amino acid residue strength including 6 residues of histidine. A polypeptide with a single histidine tag is expressed Is done.

Example 2 Preparation of YdcE from B. subtilis 168 strain, All 3211 polypeptide from Nostoc sp. PCC7120 strain, EF0850 polypeptide from E. faecalis V583 strain Expression vectors pET-ydcE and pET obtained in Example 1 — E. coli BL21 (DE3) strain (Novagen) transformed with all3211 or pET—EF0 85 for expression E. coli pET—ydcEZBL21 (DE3), pET—all321 lZBL21 (DE3) Pleasure—ET0 Got. Incubate pET—ydcE / BL21 (DE3), pET—all3211 / BL21 (DE3) or pET—EF0850 / BL21 (DE3) in 5 ml LB medium containing 100 μg / ml ampicillin at 37 ° C. When OD600nm = 0.6, IP TG (manufactured by Takara Bio Inc.) was adjusted to a final concentration of ImM to induce polypeptide expression. Two hours after the start of induction, the culture was terminated, and the cells were collected by centrifugation. 300 1 lysis buffer (50 mM NaH PO, 300 mM NaCl, 10 mM imida

twenty four

After suspending in sol, pH 8.0), the cells were crushed using an ultrasonic crusher (Handy sonic, manufactured by Tommy). 20 μl of Ni-NTA agarose (manufactured by Kagen) was added to the supernatant collected by centrifugation, and left at 4 ° C for 30 minutes. The precipitate collected by centrifugation was washed with 100 1 wash buffer (50 mM NaH PO, 300 mM NaCl, 20 mM imidazole, pH 8

twenty four

0) was washed twice. 20 1 elution buffer (50 mM NaH PO,

twenty four

300 mM NaCl, 250 mM imidazole, pH 8.0) was suspended and centrifuged, and the supernatant was collected. The same elution procedure was repeated two more times to obtain a total of 60 1 samples containing YdcE, A113211 and EF0850 polypeptides. A part of this sample was subjected to SDS-PAGE to confirm that it contained a polypeptide of the expected size. The protein concentration in the sample was about 50 ngZ μ 1.

Example 3 Identification of base sequence specificity using oligoribonucleotide as substrate

To examine the nucleotide sequence specificity of the ribonuclease activity of the YdcE, A113211 and EF0850 polypeptides obtained in Example 2, oligoribonucleotides were synthesized and subjected to cleavage assay.

[0041] Nine types of oligoribonucleotides whose base sequences were shown in SEQ ID NOs: 13 to 21 were synthesized as substrates. 10 M oligoribonucleotide, lOngZ wl YdcE obtained in Example 2, A1132 11 or EF0850 polypeptide, 51 mM reaction solution consisting of 10 mM Tris—HCl (pH 7.5) was incubated at 37 ° C. for 30 minutes. The reaction product was subjected to 20% denaturing acrylamide gel (20% acrylamide, 7M urea, 0.5 XTBE buffer) electrophoresis, stained with SYBR GREE N II (manufactured by Takara Bio Co., Ltd.), and then fluorescence image analyzer FMBIOII Multi view (Takara Bio). Fluorescence images were analyzed using a Table 1 shows the state of cleavage of each oligoribonucleotide.

As for the cutting situation, complete cutting was indicated by +++, partial cutting by ++, very weak cutting by +, and complete undissolved by one. Furthermore, based on the presence or absence of cleavage of each oligoribonucleotide and the strength of cleavage, the base sequences around the cleavage sites were compared to evaluate the specificity of the sequences. The results are shown in Table 2.

[0042] From the above results, it is clear that the YdcE polypeptide and the EF0850 polypeptide cleave RNA by preferentially recognizing the 5'—U / A CAU 3 sequence (Z indicates the cleavage site). Became. It was also found that A113211 polypeptide cleaves RNA by preferentially recognizing the 5'-UZACA-3 'sequence. MazF has been reported to cleave 5'-N / ACA-3 '(N is any ribonucleotide) (Non-Patent Documents 7 and 8), but YdcE polypeptide and EF0850 polypeptide are both ACA. It was revealed that the sequence further having a U residue at the terminal was strongly recognized, and that the A113211 polypeptide strongly recognized a sequence having a U residue on the 5 ′ side of ACA. In other words, it was revealed that these polypeptides are endoribonucleases having a base sequence specificity different from that of Maz F.

[0043] [Table 1]

Name Base sequence and cleavage site Cleavage

(/ Indicates the cut site) YdcE A113211 EF0850 mazG30 UAAGAAGGAGAUAU / ACAUAUGAAUCAAAUC 10th 10th 10th

ABC020 UGCGUUCCUGU / ACAUAACCUU +++ +++ +++

MRI027 GGGGCUCGCCUU / ACAAGCGAUUGGG

ABC019 GGUUAUGU / ACAGGAACGCAUU +++

MRI021 GAGUCGUGGGCGU / ACUUUAUGGGGC

MRI005 AGACUCU / ACACUCUAUACUCUAAAA +

M20 GGCUUAGAACAUGAAUCCGG

MRI022 GGGUCCAUUGUUUCUUAUAUAAAGGUUAGG

ABC008 AUUUGAGAUCAUAUUCAUAUU

Cutting indication +++ indicates complete cutting, ++ indicates partial cutting, + indicates very weak cutting.

[0044] [Table 2]

Name Base sequence Strength of cleavage

-1 1 2 3 4 YdcE A113211 EF0850

mazLrjO U A C A U +++ +++ +++

ABC020 U A C A U +++ +++ +++

MRI027 U A C A A

ABC019 U A C A G +++

MRI005 U A C A C +

MRI021 U A C U U Ten Ten

M20 A A C A u

MRI022 U C C A u

ABC008 U U C A u

MRI022 U A u A u

Cut site: The cut site is cleaved between -1 and eleven.

Example 4 Cleavage assembly using tRNA as substrate

In order to examine the ability of the YdcE, A113211 and EF0850 polypeptides obtained in Example 2 to cleave tRNA, cleavage was performed using tRNA synthesized by in vitro transcription as a quality.

The base sequence information of tRNA40 derived from Deinococcus radiodurans having an anticodon (UAC) corresponding to the amino acid parin was obtained from The Genomic tRNA Database (http: ZZlowelab. Ucsc. EduZGtRNAdbZ). tRNA40 DNA sequence with T7 promoter sequence to synthesize tRNA by in vitro transcription The DNA shown in SEQ ID NO: 22 having a sequence and its complementary sequence DNA were synthesized with a DNA synthesizer to prepare a 92 bp double-stranded DNA fragment. Using this DNA fragment and in vitro transcription kit (manufactured by Takara Bio Inc.), in vitro transcription was performed according to the instruction manual of the kit. The resulting reaction solution was treated with DNasel (manufactured by Takara Bio Inc.) at a final concentration of 0.5UZw 1 for 30 minutes at 37 ° C, and then treated with phenol Z chloroform, chloroform, isopropanol precipitation, and RNA. Was purified. The RNA precipitate was dissolved in sterile distilled water. By this operation, the 75-base tRNA40 shown in SEQ ID NO: 23 was obtained. This tRNA40 contains 10 X Annealing Buffer (lOOmM Tris—HCl, pH 8.0, 500 mM CH COOK, lOmM EDTA) included in the kit in an lZlO capacity.

Three

After dissolving in sterilized distilled water and allowing to stand at 75 ° C for 5 minutes, the temperature was lowered to 25 ° C over 30 minutes to form a higher-order structure of tRNA40. Using 62.5 ng of this tRNA40, the same reaction solution as in Example 3 was prepared, and after cleaving with YdcE, A113211 or EF 0850 polypeptide obtained in Example 2, 10% denaturing acrylamide gel electrophoresis was performed. The presence or absence of cutting was evaluated. As a result, there was no change in the 75 base tRNA40 in YdcE and EF0850 polypeptides. On the other hand, A113211 showed that 75-base tRNA40 was degraded into 33-base and 42-base, and cleavage occurred at the U / AC A site in the anticodon loop of tRNA40. From this result, it was shown that A113211 polypeptide has an activity capable of cleaving tRNA containing anticodon UAC. Since an A residue continues on the 3 ′ side of the site in tRNA40, it was speculated that the site could not be cleaved with YdcE and EF0850 polypeptides.

Industrial applicability

[0046] The present invention provides a novel sequence-specific endoribonuclease. Since the enzyme can recognize and cleave specific sequences in RNA, it can analyze RNA molecules.

It is useful for preparation of RNA fragments, cell control through RNA cleavage in cells (for example, inhibition of protein production), and the like.

Sequence listing free text

[0047] SEQ ID NO: 7; PCR primer ydcE— F to amplify a DNA fragment encoding YdcE prote in. SEQ ID NO: 8; PCR primer ydcE-R to amplify a DNA fragment encoding YdcE prote in.

SEQ ID NO: 9; PCR primer all3211— F to amplify a DNA fragment encoding A113211 p rotein.

SEQ ID NO 10; PCR primer all3211-R to amplify a DNA fragment encoding A113211 protein.

SEQ ID NO 11; PCR primer EF0850— F to amplify a DNA fragment encoding EF0850 protein

SEQ ID NO 12; PCR primer EF0850— R to amplify a DNA fragment encoding EF0850 protein

SEQ ID NO: 13 Oligoribonucleotide mazG30.

SEQ ID NO: 14 Oligoribonucleotide ABC020.

SEQ ID NO: 15 Oligoribonucleotide MRI027.

SEQ ID NO: 16 Oligoribonucleotide ABC019.

SEQ ID NO: 17 Oligoribonucleotide MRI021.

SEQ ID NO: 18 Oligoribonucleotide MRI005.

SEQ ID NO: 19 Oligoribonucleotide M20.

SEQ ID NO: 20 Oligoribonucleotide MRI022.

SEQ ID NO: 21 Oligoribonucleotide ABC008.

SEQ ID NO: 22 DNA to transcribe Deinococcus radiodurans tRNA 40.

SEQ ID NO: 23 RNA molecule partially equal to RNA sequence of Deinococcus radi odurans tRNA 40.

Claims

The scope of the claims

[1] An amino acid sequence described in SEQ ID NO: 1, 2, or 3 in the sequence listing, or an amino acid sequence having at least one deletion, addition, insertion or substitution of one or more amino acid residues in the sequence And a polypeptide having sequence-specific endoribonuclease activity.

[2] A nucleic acid encoding the polypeptide according to claim 1.

[3] The nucleic acid according to claim 2, which has the base sequence described in SEQ ID NO: 4, 5, or 6 in the sequence listing.

[4] A nucleic acid that is capable of hybridizing to the nucleic acid of claim 2 or claim 3 under stringent conditions and encodes a polypeptide having sequence-specific endoribonuclease activity.

[5] Recombinant DNA comprising the nucleic acid according to claim 2 to 4 and shear force 1.

[6] A transformant obtained by transformation with the recombinant DNA according to claim 5.

[7] A step of culturing the transformant according to claim 6, and a sequence-specific method in the culture.

The method for producing a polypeptide according to claim 1, comprising a step of collecting a polypeptide having RNA cleavage activity.

[8] A method for producing a single-stranded RNA degradation product, comprising a step of allowing the polypeptide of claim 1 to act on single-stranded RNA.

[9] A method for degrading single-stranded RNA, comprising the step of allowing the polypeptide of claim 1 to act on single-stranded RNA.