JPS6317434B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention resides in TransferRNA (guanosine-2') methyltransferase. Methyltransferase is also called transmethylase or methyltransferase, and is an enzyme that methylates amino groups, hydroxyl groups, and thiol groups using S-adenosylmethionine, betaine, dimethyltetine, etc. as a methyl group donor. Various specific enzymes are known, but 5'...-G-G-...3' (G represents guanosine) in the D loop of Transfer RNA (hereinafter referred to as tRAN) .
An enzyme that specifically methylates guanosine (which has the same meaning below) has not been isolated. The present inventors isolated and purified various thermostable enzymes from the highly thermophilic bacterium Thermus thermophilus , and conducted research. We succeeded and arrived at the present invention. That is, the gist of the present invention is to provide a transition R.N.
A (guanosine-2') methyltransferase (A) Action: 5' in the D loop of RA...
-G-G-...3' (in the formula, G represents guanosine)
The guanosine on the 5' side of the nucleotide sequence shown is methylated to 2'-O-methylguanosine in the presence of a methyl group donor (b) Substrate specificity 5'...-G-G in the D-loop −…3′ (in the formula, G
(denotes guanosine)) Acts on the transition RNA with the nucleotide sequence shown by (c) Optimum pH 6.6-7.4 (d) Stable pH range 5.5-9.3 (37℃, 4 hours) (e) Molecular weight 21000 (by gel filtration method). The present invention will be explained in more detail below. The physicochemical properties of the enzymatic transfer RA (guanosine-2') methyltransferase of the present invention are as follows. (b) Effect The guanosine on the 5' side of the nucleotide sequence shown by 5'...-G-G-...3' in the D-loop of tRNA is treated with a methyl group donor such as S-adenosyl-L-methionine. methylation to 2'-O-methylguanosine. Here, the D-loop means the loop closest to the 5' end and always containing dihydrouridine. tRNA cloverleaf type 2
It is thought that it acts by recognizing the following structure or L-shaped three-dimensional structure. As an activator
Mg ⁺⁺ ions are used. (b) Substrate specificity Acts on tRNA that has the nucleotide sequence 5'...-GG-...3' in the D-loop. Specifically, the enzyme tRNA ^Phe , Escherichia coli tRNA ^Met _f ,
tRNA ^Met _n , tRNA ^Phe , tRNA ^Ile (here,
Phe is phenylalanine, Met is methionine,
f is formylated, m is non-formylated, Ile is isoleucine), etc., and acts on guanosine that is already methylated in the D loop, i.e.
5'...Gm-G-...3' (Gm indicates 2'-O-methylguanosine) tRNA with a nucleotide sequence, such as E. coli (tRNA ^Tyr (Tyr
indicates tyrosine) Various types of highly thermophilic bacteria
tRNA is rarely methylated. (c) Optimal PH 6.6-7.4 (d) Stable PH range 5.5-9.0 (37℃, 4 hours) (e) Molecular weight 21000 Sephacryl S-200
(Sephacryl is a gel filtration agent manufactured by Pharmacia Fine Chemicals, trademark) by a gel filtration method. (c) The activity at the optimum temperature of 70 to 72°C is about 5 to 7 times that at 50°C. Next, the method for producing the enzyme of the present invention will be explained. The enzyme of the present invention is obtained by crushing the cells of a bacterium belonging to the genus Thermus and collecting the crushed liquid. Bacteria belonging to the genus Thermus used as an enzyme source include Thermus thermophilus . The strain includes Thermus thermophilus HB27. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Accession number: Microbiological Research Institute No. 5160), and has the following mycological properties. Morphology: Length 3 μm x Width 0.5 μm, rod-shaped, non-sporulating, Gram negative, culture state Almost single-cell colony: convex, circular, orange-yellow, diameter 2~
Growth status in liquid medium containing 8mm yeast extract and pebtone: A thin film forms on the surface and becomes cloudy. Growth temperature: below 83°C Sodium chloride concentration: Growth up to 1% DNA: G+C = 67.6-69.6% Auxotrophy: Bithion biochemical properties NO ₃ reduction: Minus production of indole: Plus production of acid and gas from sugars: Glucose: Plus (large amount) Galactose: Plus (large amount) Lactose: Plus (slight amount) Sugar: Plus Maltose: Plus (large amount) Starch: Plus Enzyme separation and purification method Thermus Thermophilus
To explain specifically the case of HB27, first, the strain was mixed with 0.5% peptone, 0.4% yeast extract, and 0.2% yeast extract.
% sodium chloride, 0.1% glycose and 0.05
Using a medium consisting of % inorganic salt vitamin solution
The cells are aerated at 80° C. for 5 to 6 hours, collected at around 300 ml, and the resulting cells are crushed by a conventional method, for example, by alumina grinding. A buffer solution is added to the crushed solution for extraction treatment, bovine pancreatic deoxyribonuclease is added to the extract and reacted, and the supernatant is taken.
Diethylaminoethyl cellulose (hereinafter abbreviated as DEAE cellulose), Phoscellulose (hereinafter abbreviated as P-cellulose), then carboxymethylated Cefatex (Sefatex is a trademark of Pharmacia, hereinafter,
The mixture is sequentially passed through a column of CM-Sephadex) and separated and purified by column chromatography. As explained in detail above, the enzyme of the present invention is
There is no example of an enzyme that specifically methylates the 5'...-GG-...3' guanosine in the D-loop of tRNA, and such an enzyme has been stably isolated. This enzyme is useful as a specific enzyme reagent in genetic engineering and biochemical research. This will be explained in more detail by way of examples below. In addition, _A260 in Examples is the absorbance at 260 nm. Example 1 (1) Cultivation of Thermus thermophilus HB27 Thermus thermophilus HB27 was grown in 0.5% peptone, 0.4% yeast extract, 0.2% sodium chloride,
0.1% glucose and 0.05% inorganic salt vitamin solution (25 g MgCl ₂ 6H ₂ O, 5 g in 1 water
_CaCl2 , _2gMnSO4・_6H2O , _0.5gZnSO4・
_7H2O , _{0.5gH3BO3 , 5gFeCl3}・_6H2O , 15
_mgCuSO4 , _25gNaMoO4・_2H2O , 50mg
_CoNO3・_6H2O , _20mgNiNO3・_6H2O , 80mg pyridoxane, 10mg folic acid, _0.5mlH2SO4 , 10mg thiamine pyrophosphate, _40mg riboflavin, 80mg nicotinamide, 80mg para-aminobenzoic acid, 10mg
Bithion, 0.4mg cyanocobalamin, 80mg pantotheic acid, 20mg lipoic acid, 200mg inositol,
5-6 at 80℃ using a medium containing 50mg choline, 50mg orotic acid, 100mg spermine).
The cells were aerated and cultured for an hour, and the bacteria were collected after about 300 hours.
We were able to obtain 80 to approximately 400 g of HB27 bacterial cells. (2) Enzyme separation and purification All operations were performed at 4°C. (A) Enzyme extraction Approximately 1 kg of HB27 bacterial cells obtained as above
An equal amount of alumina was added to the mixture and ground for about 1 hour. To this, add 2 standard buffers (0.01M,
Tris−HCl (PH7.5) −0.01M MgCl ₂ −0.05M
The enzyme was extracted by adding KCl-6mM β-mercaptoethanol (same composition shown below). The extract was centrifuged at 10,000 g for 20 minutes to separate the alumina and cell debris into a precipitate and a supernatant, and the precipitate was extracted again with standard buffer 1 and centrifuged to obtain a supernatant. The supernatants from the two centrifugations were combined, 1 μg/ml of bovine pancreatic deoxyribonuclease was added, and the mixture was left for 15 minutes. This solution was subjected to ultracentrifugation at 105,000 g for 3 hours, and the resulting supernatant was used as a crude enzyme extract (2.5). (B) DEAE-Cellulose Column Chromatography Ammonium sulfate was added to the crude enzyme solution obtained in (A) to 80% saturation, left overnight, and centrifuged at 10,000 g for 30 minutes to collect the resulting precipitate. This precipitate was dissolved in as small a volume of standard buffer as possible and dialyzed overnight against the same buffer. This dialyzed enzyme solution (1.3) was added to a DEAE-cellulose column (5.2 x 54.5 cm) equilibrated with the same standard buffer and developed with the standard buffer. The unadsorbed fraction of this column contains tRNA (guanosine-2') methyltransferase. The enzyme activity was measured as follows. E. coli unfractionated tRNA is used as a substrate, and S-adenosyl-L-methionine is used as a methyl group donor. Enzyme reaction 80μ 0.01M
Tris-HCl (PH7.5), 0.01M _MgCl2 , 5mM adenosine triphosphate, 6mM β-mercaptoethanol, 0.4OD ₂₆₀ units of unfractionated E. coli
The test is carried out by adding 20μ of enzyme solution to a solution containing tRNA and 8.1μM S-adenosyl-L-[methyl- ^14C ]-methionine (62Ci/mole). After heating at 65℃ for 30 minutes, collect 80μ of the reaction solution and apply it to a 2.5cm circular paper.
Once soaked, wash three times with ice-cold 5% trichloroacetic acid, then sequentially with ethanol-ether (1:1 v/v) and ether. After drying, the radioactivity remaining on the paper is measured using a liquid stillation counter. Under these conditions, the amount of enzyme capable of transferring 1 nmole of methyl groups per hour was defined as 1 unit. (C) P-cellulose column chromatography The unadsorbed fraction from the DEAE-cellulose column chromatography in (B) was directly added to a P-cellulose column (3.5 x 39.5 cm) that had also been equilibrated with standard buffer. The methyltransferase was then adsorbed onto this column. After washing with standard buffer, KCl concentration, 0.05M to 0.8M
Elution was performed with a linear concentration gradient (2) of
Methyltransferase activity is measured at KCl concentration of 0.44M~
A small peak appears in the 0.54M fraction (referred to as fractionation), and a large peak appears in the KCl concentration 0.64M to 0.79M fraction (referred to as fractionation), and the fraction is separated into two fractions, but the fraction is tRNA. (guanosine-2') methyltransferase activity. The enzyme solution (fraction) used here is 275ml.
The amount of enzyme was 2358 units. (D) CM-Sephadex C-50 (Sephadex is a trademark) column chromatography 275 ml of the tRNA (guanosine-2') methyltransferase fraction obtained in (C) was transferred to Diaflo PM-10.
(Diaflo is a trademark, manufactured by Amicon) membrane was subjected to ultrafiltration and concentrated to 75 ml. This solution was mixed with 0.01M Tris−HCl (PH7.5)−0.01M _MgCl2
Dialysis was performed against a buffer of -0.1M KCl-6mM β-mercaptoethanol. The generated precipitate was removed by low-speed centrifugation, and the supernatant was applied to a CM-Sephdex C-50 column (1.57 x 23.5 cm) equilibrated with the same buffer solution, and a linear concentration gradient (KCl concentration of 0.1M to 0.5M) was applied. When elution was carried out with 400ml), the methyltransferase was eluted as a well-symmetrical peak that almost coincided with the _A260 peak. The peak position is 0.26-0.31M in KCl concentration
It was hot. The obtained methyltransferase solution is 59ml.
The amount of enzyme was 1610 units. (3) Measurement of enzyme properties Measurements were made using the enzyme solution obtained in (D) of (2). (A) Optimal PH, stable PH range, optimal temperature The optimal PH is 6.6 to 7.4, and the stable PH range when treated for 4 hours at 37℃ is 5.5 to 9.0, and the stable PH range is 5.5 to 9.0, and the stable PH range is 5.5 to 9.0, and the stable PH range is 5.5 to 9.0 when treated at 37℃ for 4 hours.
The activity at 72°C was about 5 to 7 times that at 50°C. (B) Substrate specificity, action (a) Specificity for nucleotides Escherichia coli unfractionated tRNA is a good substrate, but after methylation, this tRNA is completely degraded with ribonuclease T ₂ and subjected to two-dimensional thin layer chromatography. When the methylated nucleotide is analyzed with Gm Gp (here Gm is 2′-O
-Methylguanosine, Gp indicates guanosine), and this enzyme detects guanosine on the 5' side of the 5'...-G-G-...3' nucleotide sequence.
It was shown that methylation occurs to 2'-O-methylguanosine. (b) Methylation site in E. coli tRNA ^Met _f The nucleotide sequence of E. coli tRNA ^Met _f has been determined, and it does not contain 2'-O-methylguanosine. Analysis of ribonuclease _T2 degradation products of methylated tRNA ^Met _f
As in (a), Gm Gp was generated. Next, after complete digestion with ribonuclease _T1 , DEAE-Sephadex A-25 was dissolved in the presence of 7M urea.
When the oligonucleotides were fractionated, a pentanucleotide that was not found in the ribonuclease T _1- digested product of unmodified E. coli tRNA ^Met _f was detected, which coincided with the peak of radioactivity.
This radioactive pentanucleotide was degraded into nucleotides using snake venom, phosphodiesterase, and Escherichia coli phosphomonoesterase, and then analyzed using high performance liquid chromatography. C, U, Gm, G are 2.1:
It was detected at a molar ratio of 0.78:0.75:1.0 (calculated assuming G as 1.0 mol since it is a ribonuclease _T1 degradation product), and radioactivity was present only at Gm. This revealed that the methylation site was a pentanucleotide (C, C, U) Gm Gp. Escherichia coli
Considering the structure of tRNA ^Met _f , it is possible that it has such a nucleotide composition.
Since there is only CCU Gm Gp, it was concluded that the methylation site by this enzyme is the _G19 ribose in the D loop, which is located at the 19th position from the 5'-end. This is the same methylation site found in tRNA ^Met _f of extreme thermophiles. (C) Activity against various amino acid-specific tRNAs The enzyme activity was measured under the conditions described in (2) (B) as shown in Table 1. 0.10-0.14A ₂₆₀ units of tRNA from yeast, Escherichia coli, and hyperthermophilic bacteria were added to (2).
Methylation was performed using 0.21 units of the enzyme obtained in (D). The results are shown in Table 1. (C) Molecular weight measured by gel filtration method using Sephacryl S-200
It was 21000.

【table】

[Table] As is clear from Table 1, yeast tRNA ^Phe , E. coli tRNA ^Met _f , tRNA ^Met _n , tRNA ^Phe , and tRNA ^Ile are good substrates. Meanwhile, during the D loop...Gm
Escherichia coli tRNA _Tyr and various tRNAs of extreme thermophiles, which have a modification site called Gp, are hardly methylated. As mentioned above, this enzyme is 5'...-G- in the D loop of tRNA.
It turns out that it is a methyltransferase specific to the sequence G-...3'. In other words, the cloverleaf-shaped secondary structure of tRNA seems to recognize the L-shaped three-dimensional structure. This suggests that the role of this enzyme in vivo is to methylate tRNA.

Claims (1)

[Scope of Claims] A transfer RA (guanosine-2') methyltransferase having the following physicochemical properties: (a) Effect: 5′ in the D loop of the transfer RNA...
-G-G-...3' (in the formula, G represents guanosine)
The guanosine on the 5' side of the nucleotide sequence shown by is methylated to 2'-0-methylguanosine in the presence of a methyl group donor (b) Substrate specificity 5'...-G-G in the D-loop −…3′ (in the formula, G
(indicates guanosine)) Acts on the transition RNA with the nucleotide sequence shown by (c) Optimum pH 6.6-7.4 (d) Stable pH range 5.5-9.0 (37℃, 4 hours) (e) Molecular weight 21000 (by gel filtration method)