volume 8 Number i6ieao
Nucleic Acids Research
A route to RNA with an alkylating group it the 5'-triphosphate residue
M.A-Grachev, A.A.Mustaev and S.I.Oshevslti
Novosibirsk Institute of Organic Chemistry, Siberian Division of the Academy of Sciences,
Novosibirsk 90, USSR
Received 15 May 1980
ABSTRACT
Reaction of ATP with N,N,N'-tris(2-chloroethyl),N'(p-formylphenyDpropylenediamine-1,3 (abbreviation C1*R) afforded a
Y-ester of ATP (abbreviation CLRpppA) - the product of alkylation by an aliphatic nitrogen mustard residue of ClxR. The
alkylating activity of the aromatic nitrogen mustard residue
of CIRpppA is suppressed by the electron-acceptor effect of the
p-fonnyl group. CIRpppA is a substrate of RNA-polymerase, and
affords RNA with CIRpppA-residues at the 5'-termini.
INTRODUCTION
Grineva et al.(1) have proposed in 1968 the so-called
addressed chemical modification of nucleic acids. This approach
is a kind of the affinity modification techniques, wherein
affinity of the reagent to the target is based on Watson-Crick
complementarity.
It has been found in 1974 (2) that jf-anilidate of ATP is a
substrate of E.coli RNA-polymerase. It was established later
that the substrate activity is a rather general property of NTP
Y-derivatives in this system (3-5). These findings opened the
possibility to obtain transcripts bearing various reactive
groupings at the 5'-triphosphate termini. On this basis, an
approach has been elaborated to affinity modification of RNApolymerase within the transcription complex (5).
The present communication describes the synthesis of a Y -ester of ATP with a specially designed, regulated alkylating
group attached to the 5'-triphosphate residue, the proof of the
substrate activity of this compound in the RNA-polymerase system and the preparation of RNA bearing an alkylating group at
the 5'-triphosphate terminus. Thus made is the first step to
O I R L Press Umrtsd. 1 Falconberg Court London W 1 V 6 F G . U.K.
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addressed alkylation of DNA nearby the startpoints of transcription.
MATERIALS AND METHODS
DEAE-cellulose DE-41 and phosphocellulose P-11 were purchased from Whatman (England), Sephadexes - from Pharmacia
(Sweden), anionite Lichrosorb.NHp - from Merck (ERG), disodium
ATP (98% purity) - from Reanal (Hungary), the other nucleoside-5'-triphosphates - from Serva (ERG),
[14c]-UTP - from Amersham
(England), [ Y - ^ 2 P ] - A T P - from the same firm, alkaline phosphomonoesterase of E.coli (BAPP, electrophoretically pure) - from
Worthington (USA).
Agarose-bound snake venom phosphodiesterase was prepared by
Dr.
S.Oresnkova. DNAs from phages T7 and M13 were kindly given
by Mrs.T.G.Maksimova. Determination of the specific
of core
activity
RNA-polymerase with T7 DNA according to ref.(6) gave
a value 350 units/mg,
and of
holo-enzyme - 8000 units/mg; the
preparations of these enzymes were kindly given by Mr.V.Khomov.
N,N,N'-Tris(2-chloroethyl),N'(p-formylphenyl)propylenediamine-1,3 has been synthesized in this Institute as described
by Gall et_al.(7).
UV-spectra were measured by means of a double-beam recording
spectrophotometer Specord-UV-tfTS (Carl Zeiss, GDR.); radioactivities were counted using Markll scintillation counter of Nuclear Chicago (USA) and dioxane, toluene scintillation liquors,
or by Cherenkov effect. Micro-column chromatography was performed according to ref. (8) using the double-beam microspectrophotometer designed by Kuzmin (9). Preparative liquid chromatography was performed using the equipment purchased from LKB
(Sweden); Uvicord-II was used as the UV-monitor. NMR-spectra
were measured by means of Fourier spectrometer HX-90 of Bruker
Physik (FRG) at 36.5 Mcps.
Alkylation of ATP leading to CIRpppA was performed in 50%-dimethylformamide at 40° in 0.2 M sodium borate buffer pH 8.2
at concentrations of ATP and C1,R 10 mfil. The volumes of reaction mixtures in different experiments varied between 100 and
400 jil. pH of the solution of ATP added to the reaction mixtures was adjusted to 8. The reaction was run for 1h resulting
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in a decrease of pH to 7.5. The reaction mixture was separated
by chromatography on DEAE-Sephadex A-25 (Fig.1). The fractions
of the product CIRpppA were combined and evaporated to dryness
a few times with water to remove triethylammonium bicarbonate.
The yield of CIRpppA was 25-30%.
Reduction with NaBH was performed at a concentration of NaKL
10-50 mil at a 100-fold excess relative to aldehyde groups (or
greater) in O.3M sodium borate buffer pH 7.8 for 10 min at 25°.
Reaction of reduced CIRpppA with ethylenedlamine. Reduced
CIRpppA solution was supplemented with 1M ethylenediamine.HCl
pH 9.2 to a concentration 0.5M. In 10-15h of incubation at 40°,
aliquotes of reaction mixture were diluted with 100 volumes of
water and applied to 50 jil micro-columns with DEAE-cellulose.
The separations were run as described in legend to Fig.3.
Treatment of CIRpppA with E.coli alkaline phosphatase. 0.5 units
of alkaline phosphatase was added to 0.16 b-o&o UTl"l'bs o f ft - Pj-ClRpppA in 20 jil of 0.1M Tris.HCl pH 8.4 - 0.1 M NaCl - 0.02M
MgCl_ and the mixture incubated for 60 nHn at 37° • An aliquote
of the reaction mixture was analyzed by micro-column chromatography (Pig.3d).
Hydrolysis of CIRpppA by immobilized snake venom phosphodiesterase. A 20 ul column with immobilized phosphodiesterase (3-2
activity units) was washed with 100 jil of 1M NaCl, 200 jil of
water and 100 jil of 0.01M Tris.HCl pH 8.8 - 0.0141 MgCl2- 0.2
A
260 units of CIRpppA in 10 jil of the same buffer was applied
to the column and incubated for 40 min at 37°. The column was
washed with 100 ul of water and the effluent analyzed by micro-column chromatography on anionite Lichrosorb.M^ with detection
at 260 and 360 nm. The volume of the chromatographic column was
50 ul, elution was performed with 600 jil of a linear gradient of
potassium phosphate pH 3.9 (0.005 to O.5M), the rate of elution
was 5 jil/min (Pig.4).
Substrate properties of CIRpppA in the RNA-polymerase system
were studied essentially as in ref.(2). The reaction mixture
(55 jil) contained 4-10-4M of each UTP, GTP, CTP; 3.5*1O"5M
CIRpppA; 0.5 jiCip4c]_ U T P (300mCi/mmole); 0.07M Tris.HCl pH 7.8;
0.12 M KC1; 7-10"% EDTA; 0.01M HgCl 2 ; 8 activity units of holo-RNA-polymerase; 100 jzg/ ml T7 DNA. At time intervals, 5 jil aliquotes were withdrawn, applied to pieces of '.7hatman 3L3LI paper
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and these pieces washed three times with 5% trichloroacetic
acid at 5°» with ethanol and ether, and counted in toluene
scintillation liquor. Controls were run under identical conditions, but without CIRpppA, or with 4'10~\l ATP instead of
CIRpppA. Reactions were run at 37° •
Additional purification of[^2p]- CIRpppA. Fractions corresponding to [^-32^ -ClEpppA (Flg.1) were collected, evaporated to
dryness a few times and dissolved in 150 jil 0.1M Tris.HCl pH
8.4 - 0.1M NaCl - 0.02M MgCl2. In 1 h of incubation after addition of 0.8 units phosphomonoesterase, the reaction mixture
was passed through a column with 1 ml of phosphocellulose prewashed with 0.1M potassium phosphate and with water. Elution
of the product with water was monitored by counting the radioactivity. The fractions containing[f-^Pj-ClRpppA were subjected to chromatography as described above. The product was desalted by evaporation and passed through Chelex.
Synthesis of RNA on M13 SNA template in the system containing
fy-32p] -CIRpppA. The reaction mixture (60_pl) contained 0.27
5
A 2 6 Q units of M13 DNA; 5-10~\ each DTP, GTP, CTP; 3-1O~ M
[y -?2p]-CIRpppA (O.1Ci/mmole); 0.07M Tris.HCl pH 7-8; O.7 mM
EDTA; 1mM dithiotreitol; 10mM MgClp. The reaction was started
by adding 50 jig core-RNA-polymerase. In 15 min of incubation,
O.75 ul of O.O1M ATP was added. In 60 min of incubation at 37°
the mixture was diluted with 200 jil of 0.2% aqueous dodecylsulphate (SDS), and four times extracted with phenol saturated
with 0.05M Tris.HCl pH 8. The aqueous phase after the extraction was subjected to gel-filtration on Sephadex G-50 (30 ml,
h=30 cm); the flow rate was 15ml/h; 15 drops (1ml) fractions
were collected. Fractions of the polymer peak: were combined
and subjected to alkaline hydrolysis. The yield of the transcript was 12 000 cpm (Cherenkov), see Fig.7.
Alkaline hydrolysis. The transcript containing the 5'-terminal
[Jf-32p]-ciRpppA-grouping (2000 cpm) was subjected to alkaline
hydrolysis for 4h at 40° in 0.3U NaOH. The mixture was neutralized with HC1, diluted with water to 15 ml and subjected to
chromatography under conditions described in the section
"Alkylation of ATP". 6 A 2 6 Q units of CIRpppA and 4 A 2 & 0 units
of ATP were used as carriers and markers. The result is shown
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in Fig.SA. The fractions corresponding to the major peak of
radioactivity were combined and evaporated to dryness. It will
be seen below that this product is CIRpppAp.
Dephosphorylation of radioactive CIRpppAp obtained in the previous experiment was performed by phoaphomonoesterase (1.6 a.u.)
in a reaction mixture containing along with radioactive CIRpppAp
also 5.5 A _ g 0 units of ATP and 7 A 2 6 0 units of non-labelled
ClRpppA in 200 jil of 0.1M Tris.HCl pH 8.4 - 0.1M NaCl - 0.02M
MgCl
The reaction was run for 1h at 37°. After this, chromatography revealed the pattern shown in Pig.8b. Fractions of the
main peak of radioactivity were evaporated. This peak contained
radioactive ClRpppA.
Reaction of labelled ClRpppA with ethylenediamine was performed
using the above radioactive ClRpppA as described under "Reaction
of ClRpppA with ethylenediamine", separation of the products - as described under "Alkylation of ATP". After reduction, reaction with ethylenediamine and dilution, the mixture was supplemented with 4ApgQ units of ClRpppA and subjected to chromatography (Pig.8c).
RESUI/TS
Recently Salganik et_al.(10) and independently Summerton
and Bartlett (11) have proposed a variant of the method of addressed chemical modification of nucleic acids according to
which chemically reactive groups are attached vo the base residues of a large RNA molecule. If such an RNA molecule bearing
randomly attached reactive groupings is hybridized with a complementary region of DNA, numerous chemical lesions may be introduced into a large part of the genome, e.g., into a particular gene. In order to elaborate such an approach, it was necessary to obtain special reagents containing regulatory functions so that it would be possible to "switch on" reactive groupings only after hybridization. Summerton and Bartlett (11)
have used for the regulation purpose an aliphatic ketal function. Transformation of this ketal to keto-group "switched on"
the reactivity of the adjacent alkylating function. A disadvantage of this way of regulation is the use of rather drastic
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acidic treatment for the hydrolysis of ketal.
For the same purpose, Salganik et al.(10) employed the reagent synthesized by Gall et al.(7). This reagent is a trifunctional nitrogen mustard - N,N,N'-tris(2-chloroethyl),N'-(p-formylphenyl)propylenediamine-1,3 (abbreviation C1,R):
Aliphatic alkylating groups of this reagent were used to bind
it to "addressing" RNA. The aromatic nitrogen mustard moiety
is inactive due to the electron-acceptor effect of the formyl
residue. Activation may be achieved by reduction of the formyl
group with sodium borohydride under extremely mild conditions.
The reactivity of the aromatic nitrogen mustard residue thus
increases some 1000 times.
In the present studies, we used the reagent of Gall et al.
(7) to obtain a derivative of ATP with an alkylating group of
controlled reactivity.
Synthesis of CIRpppA and Proof of Its Structure
It was decided to obtain the derivative of ATP - CIRpppA:
0
H
*C-(VN-(CH2)3-NH o
o
o
CH2m
CH2CH20-f>-0-P-0-P-0-CH2
CH C1
2
"o ~o "o k
' OH
OH
by means of alkylation of ATP with C1,R. Among other possible
routes of synthesis, this one was selected as the simplest and
the most suitable for the preparation of highly radioactive
product, although it was obvious that the yield should not
be high because of side reactions. The competition factor of
the phosphate residue at weakly alkaline pH is somewhat greater than that of the adenosine residue in alkylation of AMF
(12). The
Y-phosphate residue of ATP must have even greater
activity than the phosphate residue of AMP. Therefore it was
expected that at moderate extents of alkylation CIRpppA should
be the major reaction product in the reaction of C1,R with ATP.
The alkylation was performed in 50% dimethylformamide because of the poor solubility of C1,R in water. Weakly alkaline
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pH was maintained by borate buffer which does not react with
C1,R. The conditions were optimized on the basis of the data
of multiple-wavelength micro-column chromatography (8) on DEAEcellulose at pH 7.5. In the course of alkylation, a compound
was accumulated with properties expected for ClEpppA, i.e.,
chromatographic mobility corresponding to net charge -2 and
UV-spectrum similar to that of an equimolar mixture of G1,R and
ATP. Maximum yield of this compound (25-30%) was obtained with
equimolar amounts of reagents. Fig.1 shows the result of the
separation of the reaction mixture on DBAE-Sephadex A-25; in
this particular experiment, the reaction was run with f)(-32pj-ATP. Peak 1 corresponding to CIRpppA was evaporated to dryness
to remove triethylammonium bicarbonate, and this product, or
a similar product obtained from non-radioactive ATP, were
subjected to different analytical procedures to prove the
structure.
Fig.3a shows the result of micro-column chromatography of
non-radioactive CIRpppA. It is seen that the compound is homogeneous, and that its spectral characteristics remain constant
along the peak.
The UV-spectrum of CIRpppA is shown in Fig.2. A characteristic feature of this spectrum is the presence of two maxima
-0.7
Fig.
Separation of the products of reaction of fy-32p]-ATP
with CI3R on DEAE-Sephadex A-25. Volume of column 6 ml,
h=10cm, linear gradient of triethylammonium bicarbonate
pH 7.5, 0 to 0.5M (400 ml); 30 ml/h; 4.5 ml fractions.
A254;
concentration of eluent; -•—•- radioactivity. 1 - CIRpppA; 2 - ATP.
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Pig.2. UV-spectra in
water at pH 8.
1- CLRpppA;
2- sum of the
spectra of
C U R and ATP;
3- product of
reduction of
ClEpppA with
NaBH,,
1-10
20
2 SO
300
350
400 n m
nearby d60 and 350 nm (curve 1). Curve 2 is the calculated
spectrum of an equinolar mixture of ATP and C1,R. The similarity of these two spectra proves the presence of the formylphenyl residue (TnmHmnTn at 350 nm, cf.ref .(7))» and also the
fact, that this residue is present at an equimolar ratio with
the adenosine residue. Curve 3 in the same Figure is th_e UVspectrum of the product of reduction of CIRpppA with NaBH.; it
is known that this change of the spectrum is due to the transformation of formyl to hydroxymethyl group (7).
Figures 3 b and 3c illustrate the changes of chromatographlc
mobilities caused by reduction of CIRpppA with sodium borohydride followed by either hydrolysis (b) or reaction with ethylenediamine (c). It is seen that reduction followed by hydrolysis changes the spectral characteristics, but does not change
the chromatographic mobility. Reaction of the reduced product
with ethylenediamine which presumably gives the product of
alkylation of HH2CH2CH2NH2 by the aromatic alkylating group,
as expected, gave a compound with chromatographic mobility
greater than that of CIRpppA and corresponding to a net charge
-1 (Pig.3c). The ratio of the areas of the peaks in Pig.3c
suggests that CIRpppA contains at least 80% of intact 2-chloroethylamine residues. This method of evaluation of the content
of intact 2-chloroethylamino groups based on alkylation of
ethylenediamine has beon employed for the first time by Knorre
et al.(15): ethylenediamine is a convenient"scavenger" because
it has a large competition factor, and because it introduces
a positive charge into the product of reaction.
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400
BOO
Fig.3. Micro-column chromatography of various products and reaction mixtures. Column with DEAE-cellulose EE-41, volume
50 ul, h=50mm: linear gradient of NaCl 0 to O.3M in 7M
urea - 0.01M Tris.HCl pH 7-5 (600ul): 25ul/min; detection
at 260 (
) and 360 (
) nm. In (d), 20ul fractions
were collected.
a- CIRpppA; b- product of reduction of CIRpppA with
i
followed by Hydrolysis; c- product of reaction of reduced
CIRpppA with ethylenediamine; d- product of treatment of
CI
-32p]_ciKpppA with phosphomonoesterase. • • radioacvity.
S
To prove that CIRpppA is the product of alkylation of the
y-phosphate group of ATP, we treated the ["J-^ p] -compound with
alkaline phosphomonoesterase of E.coli. It is known that the
enzyme hydrolyzes ATP and ADP to adenosine (14), whereas ^~
-amides and Jf-esters of ATP are not attacked (2,3). It is seen
in Fig. 3d that CIRpppA remains intact on treatment with phosphomonoesterase. This result also proves that CIRpppA does not
contain any considerable admixture of compounds with unsubstituted phosphate groups, like, e.g., products of alkylation of
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ATP at the adenosine residue having the same chromatographic
mobility as CIRpppA.
It is known that "^-derivatives of NTP are cleaved by snake
venom phosphodiesterase to give NMP and substituted pyrophosphates (2). Pig.4 shows the result of the action of this enzyme
upon CIRpppA. It is seen that hydrolysis yields two products
retained by anionite. The spectral characteristics and the
chromatographic behaviour of one of these products are the
same as those of AMP. The spectral characteristics of the other
product are the same as those of CI3R, whereas the chromatographic mobility corresponds to that expected for a substituted
pyrophosphate. The structure of CIRpppA is also in accord with
the NMR-spectrum shown in Fig.5.
Hence, the above evidence proves the structure of ClEpppA
shown in its formula except for the fate of the second 2-chloroethylamino group. The chromatographic mobility of CIRpppA
and of the product of its reaction with ethylenediamine indicates that this 2-chloroethylamino group has not been transformed into a stable ethyleneimmonium cycle. It is most probable that this group has been transformed into a 2-hydroxyethylamino group as shown in the formula; it must not have survived
long contacb with water during the isolation procedures.
Substrate activity of CIRPPPA in the system of RNA-polymerase
of E.coli.
It has been found in this laboratory (2) that ATP #-anilidate is both an initiating and an elongating substrate of RNA-
A
a
O5
A
OS
1
A
A
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b
2
A,
200
400)4
Fig.4. Micro-column chromatography of ClRpppA(a)
and of the products of
its phosphodiesterase
hydrolysis (b) on anionite Lichrosorb.NH-.
Volume of column 50 pi,
h=50mm; linear gradient
of KH2PO4 pH 3.9, 0.005
to 0.5M (600ul); 5ul/min.
1- AI£P; 2- CIRpp.
•••A240;
Nucleic Acids Research
Pig.5. ?1P-NMR spectrum of
CIRpppA, 0.01M in water
pH 10, passed through.
Chelex.
36.43Mcps;spin-spindecoupling; 30°;
2000 scans; internal
standard 85% H3PO4.
Cf.ref. (13).
23 6 ppm
-polymerase of E.coli. Later, Armstrong and Eckstein (3) have
found that Jf-esters of nucleoside-5'-triphosphates also exhibit substrate activity, and Yarbrough (4) made a similar finding for an ATP amidate with a bulky substituent - 1-aminonaphthalene-5-sulfonate - in the Jf-position. These data along
with our unpublished findings on the substrate activity of many
other analogues of NTP prompted that CIRpppA should also be active as a substrate of E.coli RNA-polymerase.
Pig.6 shows kinetics of the incorporation of radioactivity
into acid-insoluble fraction during RNA-polymerase-catalyzed
synthesis on T7 DNA template in reaction mixtures containing
[14C]-UTP, GTP, CTP, ATP (curve 1); [1^C]-DTP, GTP, CTP, CIRpppA
(curve 2); and f^c]-UTP, GTP, CTP (curve 3). It is seen that
CIRpppA stimulates the incorporation of radioactivity when
taken as the fourth substrate instead of ATP. It is noteworthy
that special care was taken to avoid any admixture of ATP in
CIRpppA. Micro-column chromatography failed to detect any admixture, although it would be reliably detected if present in
an amount greater than 0.5%. On the other hand, consumption of
Pig.6. Kinetics of the synthesis
of RNA by E.coli DNA-dependent
RNA-polymerase on T7 DNA
template.
Substrates:
1-P4C]UTP,
GTP,CTP,ATP
2- [1^C]UTP, GTP,CTP,CIRpppA
3- [1^C]UTP,GTP, CTP
Concentrations:
ATP,UTP,CTP,GTP - 0.4mH each
CIRpppA- 3.3>mLl
5 ul a l i q u o t e s
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ClEpppA in the synthesis of RNA was about 3%, as calculated
from the data shown in Pig.6. Therefore CIRpppA is an elongating substrate of E.coli
RNA-polymerese.
However, the aim of the present studies was also to prove
that CIRpppA also acts as an initiating substrate, i.e., that
it is incorporated into the 5'-terminus of newly-synthesized
RNA. To make the corresponding experiments, we prepared [ Y - ^ P ] -CIRpppA. This substrate was used to synthesize RNA on single-stranded M13 DNA template in a combination with GTP, CTP, UTP.
The product of this reaction, presumably a heteroduplex (15) of
newly-synthesized RNA with template DNA, was isolated by gel-filtration (Fig.7) and subjected to alkaline hydrolysis. The
hydrolysate was chromatographed on DEAE-sephadex in the presence of ATP as carrier and marker. It is seen in Fig.8a that
the major peak of radioactivity is eluted immediately after ATP.
This product was treated with phosphomonoesterase and co-chromatographed with non-radioactive CIRpppA. Obviously, data shown in
Fig.8 prove that the ClRpppAp-residue is present at the 5'-terminus of RNA synthesized from CIRpppA and three other substrates,
i.e. that CIRpppA is an initiating substrate of E.coli RNA-polymerase.
The substance of peak 2 (Fig.8b) was treated with NaBIfy followed by ethylenediamine. After dilution, the mixture was supplemented with CIRpppA and subjected to chromatography. It is
seen in Fig.8c that some 6 0 % of the radioactivity is eluted
with the product of reaction of reduced CIRpppA with ethylene-
Fig.7. Gel-filtration of the reaction mixture after synthesis
of RNA from [-y-32p]-CIRpppA and CTP,GTP,UTP on M15
DNA template.
*-?6O> ~~*
* — radioactivity.
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Fig.8. Chromatography
on DEAE-Sephadex, conditions as in Fig.1.
a- alkaline hydrolysate,
see text.
b- product of peak 3
(Fig.8a) treated
with phosphomonoesterase. Carrier- CLRpppA.
c- peak 2 (Fig.8b)
reduced with NaBIfy
and treated with
ethylenediamine;
CIRpppA added after
dilution.
?54 acHviE£
Carriers identified
by UV-spectra
SO Ir
diamine. Hence, a large proportion of the "switched-out" alkylating groups survive even drastic alkaline hydrolysis.
DISCUSSION
Hence, the above evidence proves that the substrate activity
of ClEpppA in the RNA-polymerase system makes it possible to
obtain transcripts which bear a latent alkylating function at
the 5'-triphosphate end. This group may be activated under very
mild conditions by sodium borohydride treatment. According to
preliminary data obtained in this laboratory, such "alkylating
transcripts" may be used for addressed alkylation of template
DNA nearby the startpoints of transcription. It seems to us
that selective modification of DNA at the promoter regions
might become a useful tool for their structure-functional studies in vivo and in vitro. However, the proof of the possibility of addressed alkylation in this system will be a topic of
another publication.
Alkylating transcripts might be used, presumably, also to
study the conformation of pro-mRNA and of its changes during
processing, by analogy with the use of an alkylating reagent
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attached to the 3'-end in a study of the conformation of tRNA
in solution (16). The alkylating function is very convenient
to this end, because under appropriate conditions it induces
cleavages of the polynucleotide chain whose positions may be
easily found by modern rapid sequencing procedures.
The ClR-residue is rather huge (length about 15A) and hydrophobic. Hence, the substrate activity of CIRpppA sheds some
light on the topography of the transcription complex - it suggests that the triphosphate residues of both the initiating
NTPs and the elongating NTPs reside close to the outer surface
of the protein globula.
Finally, the synthesis of CIRpppA described above suggests
that alkylation of the phosphate residue may become a general
route to many
y-derivatives of NTPs which may be used as sub-
strate analogs with reactive and reporter groupings.
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1 Grineva.N. [. , Zarytova, \T.F., Knorre .D.G. (1968)Izv.Sib.otd.
Akad.Nauk SSSR, ser.khim.nauk, vip.5, 118-124
2 Grachev,M.A., Zaychikov.E.F.(1974) FEBS Letters 49 163-166
3 Armstrong,V.W., Eckstein,F.(1976) Eur.J.Biochem.70. 33"38
4 Yarbrough.L.R. (1978) Biocem.Bioph.ysiRes.Comm. 81 35-41
5 Sverdlov,E.D. ,Tsarev,S.A., Modyanov.N.N., TJTpkTn,V.M.,
Grachev,M.A. , Zaychikov.E.F. , Pletnev.A.G. (1978) Bioorp;.
khimia. 4, 1278-1280
6 Gonzalez,N., Wiggs.J., Chamberlin.M.J.(1977) Archives Biochem.Biophya. 182 404-408
7 Gall, A.A., Kurb~atov,V.A., Mustaev.A.A., Shishkin.G.V.(1979)
Izv.Sib.otd.Akad.Nauk SSSR, ser.khim.nauk, vip.2, 99-104
8 Grachev,M.A.(1973) in gitramicroanalysis of Nucleic Acids,
eds.Knorre D.G. , i/enkstern,T.V., pp. 104-122, Nauka, Moscow,
9 Kuzmin, S.V. ibid., pp.95-104
10 Salganik.R.I. , Dianov.G.L., Kurbatov.V.A. , Shishkin.G.V.,
Gall,A.A.(1978) Dokl.Akad.Nauk SSSR 239, 217-219
11 Summerton.J., Bartlett.P.A.(1978) J.Mol.Biol. 122, 145-162
12 Grineva.N.I., Knorre,D.G., Kurbatov,/.A.(1971) Izv.Sib.otd.
Akad.Nauk SSSR, ser.khim.nauk, vip.2, 107-111
13 Zarytova,V.F., Knorre,D.G., Kurbatov,V.A., Lebedev.A.V.,
Samukov.V./., Shishkin.G.V.(1975) Bioorg..khimia 1 793-798
14 Heppel.L.A., Harkness.D. , Hilmoe,R.J.(1962) J.Biol.Chem.237
841-846
15 Chamberlin.M.J.. Berg,P. (1964) J.Mol.Biol.8, 297-313
16 Grachev,M.A., Rivkin,U.I.(1975) Nucleic Acid Res.2,1237-1260
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