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On the Secondary Structure of Soluble Ribonucleic Acids.

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the following, experiments are described which prove iPA to
be N(6)-(y,y-dimethylallyl)adenosine ( I ) .
The high-resolution mass spectrum of iPAr5l showed its
elemental composition to be Cl~H21N504.The abundance
of ions containing five nitrogen atoms and no oxygen,
particularly CsHsN5 and CloH13N5, indicated the presence
of a substituted adenine pentoside. Nitrogen-free ions
containing four oxygen atoms, particularly C5H904. show
that all of the oxygen atoms are in the sugar moiety. It
follows that iPA is an adenosine derivative substituted by
five carbon atoms. The presence of ions of the type C,HmN
up to (CzHgNH) i- indicates an A7(6)-C5-alkenyl or cyclopentyl adenine derivative, which can eliminate all 5 carbon
atoms o n electron impact. A distinction between the two
could be made by consideration of the fragments formed
from the C10H13N5 ion, which represents the free adenine
derivative formed [61 on electron impact.
The low abundance of 8 C8H8 or 9N5 ion as contrasted t o that
of Ns-ions containing 6,7, or 9 carbon atoms implies that
loss of two carbon atoms (as C2Hs or C2H4) from the side
chain is much more difficult than the loss of one, three, or
more carbon atoms. This behavior excludes a cyclopentyl
substituent and suggests an isopentenyl group. The position
of the double bond was clarified by N M R spectroscopy[71.
The spectrum shows the signals of the two aromatic adenosine
hydrogens unchanged at 8.1 and 8.3 ppm (each of relative
intensity 1). The signal at 8.1 ppm is superposed by the
resonance of one amidine hydrogen, which exchanges slowly
with deuterium (relative intensity 1). It therefore may be
concluded that this nitrogen (at the 6-position) carries an
additional substituent, i.e. the isopentenyl group. The signals
of the 1’- and 5’-hydrogens of the ribose moiety appear at
5.94 pprn (intensity 1) and 3.84 ppm (intensity 2), respectively,
as they d o in adenosine. The region between 4.1 and 5.6 ppm
cannot be analysed because of strong absorption by the
solvent. A sharp signal at I .73 ppm (intensity 6) proves the
presence of a 2,2-dimethylvinyl group in the isopentenyl
side chain.
The acid conversion product A has the composition C10H13N5
according to mass spectrometry. The difference in abundance
of the C,H,N5 ions in this spectrum as compared to that of
;PA, particularly the lower abundance of (CsHsNs)+ in the
former, suggests the absence of an open-chain substituent
attached to N(6) only. Acid-catalysed cyclization with the
adenine ring would account for this product. The product B
thus contains
has an elemental composition C I O H ~ ~ N
one molecule of water more than the base of the iPA. The
high abundance of a C3H70--ion requires hydration of the
double bond to produce a C(OH)(CH,)* grouping; this is
also corroborated by ions (C9H12N50 and C7HsN5) representing the loss of a methyl- or dimethylcarbinol group.
Simultaneously with this work, iPA was isolated from unfractionated tRNA, elucidated, and synthesized [81.
I t is interesting to note that the plant growth hormone zeatin
is a n N(6)-(..-methyl-y-hydroxymethylallyl)adenine~’J~ and
that N(6)-(y,y-dimethylallyl)adenine
promoting activityrlol.
also shows growth
Received: M a y Sth, 1966
[Z 217 JE]
G er man version: Angew. Chem. 78, 600 (1966)
[I] Communication X on Serine-specific Transfer Ribonucleic
Acids. - Communication IX: G. H. Zachau, D . Diitting. and
H . Feldmann in: Structure and Function of Genetic Elements.
Symposium of the Federation of European Biochemical Societies,
Warsaw 1966. Academic Press, in press.
[2] Abbreviations: tRNA, transfer ribonucleic acid; iPA, N(6)isopentenyladenosine or N(6)-(.{,y-dimethylaIlyl)adenosine.
[3] H. G. Zachau, D. Diitting, and H . Feldinanlt, Angew. Chem.
78, 392 (1966); Angew. Chem. internat. Edit. 5, 422 (1966).
[4] Further details on iPA: H . Feldnzanir, D. Diitting, and H . G.
Zacharr, to be published.
[5] The spectrum was obtained by vaporizing part of the eluate
from a paper electropherogram containing about 10-20 pg iPA
directly into the ion source of a CEC 21--110 double-focusing
mass spectrometer; K . Bietnaizn, P. Botiitiier, and D. Desideri.),
Tetrahedron Letters 1964, 1725.
[6] K. Biernann and J. A. McCloskey, J . Amer. chem. SOC.84,
2005 (1962).
[7] The spectrum was obtained from 300 pg iPA in DzO with an
A-60 Varian-Spectrometer connected to a C-1024 Time Averaging
[8] R. H. Hall, M. J . Robins, L. Stasirtk, and R.Thedford, J .
Amer. chem. SOC.,in press.
[9] D . S. Letham, J. S. Shannon, and I. R. McDonald, Proc.
chem. SOC.(London) 1964, 230.
[lo] H . Q. Hainzi and F. Skoog, Proc. nat. Acad. Sci. U.S.A. 51,
76 (1964).
On the Secondary Structure of Soluble
Ribonucleic Acids
By Dip].-Chem. H. Doepner, Dr. H. Seidel,
and Prof. Dr. F. Cramer
Max-Planck-Institut fur Experimentelle Medizin,
Gottingen (Germany)
N-Oxidation of adenosine units in polynucleotides with monoperoxyphthalic acid at p H 7.0 is inhibited by base pairing,
a fact that can be used[” to obtain information on the
secondary structure of s-RNA[ZJ. We have applied this
method to R N A from various sources isolated by different
methods. s-RNA from brewer’s yeast (AMP 19.8 %, UMP,
24.5 %, G M P 28.2 %, CMP 27.5 %, and terminal A 47 X of
the theoretical value) and s-RNA from Escherichia coli B
(AMP 20.5 %, U M P 18.2 %, G M P 30.2
C M P 31.1 %. and
terminal A 34.0 of the theoretical value) were examined in
the native state and after heat denaturation and cooling. The
results were interpreted as described previously [I]. The
quotient “232 ms’E259 ml* served as a measure for the degree
of oxidation. The average content of oxidized AMP units per
molccule was determined from this quotient by means of il
s - R N A froin
brewer’s ycaxt
heated at:d
heated a n d
Final degree of oxidation(rzJr/czss) A M P units ohidized
per molecule
per molecule in single-strand regions
(18 ”<)
Av. no. of nucleo:ides per m o l : x u k
A v . no. of nucleotide units
( 2 3 %I
(30 7;)
A \ . n o . of base pairs per inolecule
Hyperchromicity of the untrentc.l
(0.4 M phosphate; 0.01 h.1 M g ” )
Airgew. Cliem. iiiterntrt. Edit.
Vol. 5 (1966)
/ No.6
31 5
calibration curve. By addition of the remainder of the bases
in the ratios determined by analysis, the average total number
of nucleotides present per molecule in single-strand regions
was then derived.
The low oxidizability of s-RNA from brewer’s yeast compared to that of the s-RNA from E. coli B is probably due
to its higher content of U M P and is in accordance with its
stronger hyperchromicity 121. If s-RNA is heated for a prolonged period above its “melting temperature” T, [*] and
then gradually cooled, the oxidizability of both preparations
is significantly higher; the hyperchromicity also decreases
with this treatment.
Subtraction of the average number of nucleotide units present
per molecule in the single-strand regions from the average
total number of nucleotides per molecule[31 gives the number
of nucleotides occurring in double-strand regions (and hence
the number of paired bases), which when plotted against the
values for the hyperchromicity gives an almost linear dependence between about 15 and 40 % hyperchromicity.
Data for the N-oxidizability and hyperchromicity of s-RNA
from baker’s yeast obtained previously under different conditions[ll exhibit this same dependence within the limits of
error. Thus, a direct relationship between the hyperchromicity of s-RNA and the degree of base pairing is established.
The latter can be determined by means of N-oxidation.
s-RNA has presumably a variable secondary structure which
depends strongly upon the Mgz+ concentration and perhaps
on other factors as well. A means is now available for
determining the extent of base pairing even with minute
amounts of substance by measurements of hyperchromicity.
Received: May Znd, 1966
[Z 218 IE]
German version: Angew. Chem. 78, 601 (1966)
[I] H. Scidzl and F. Crnnrer, Biochim. Biophysica Acta 108, 367
[2] Abbreviatims: A = adenosine. - s-RNA = soluble ribo-
nucleic ecid. - AMP = adenosine monophosphate. - UMP =
uridinc monophosphate.
CMP = cytidine monophosphate.
G M P = guanosine monophosphate.
Tm = t h e temperature at
half the total extinction increment. - Hyperchromicity is the
increase in extinction on thermal breakdown of ordered macromolecular structures.
[3] G. L. Brown and G. Zubny, J. molec. Bjol. 2, 287 (1960);
J. E. M . Midgley, Biochim. biophysica Acta 108, 340 (1965);
T. Lindnhl, B. B. Henley, and J . R . Fresco, J. Anier. ch-m. Soc.
87, 4961 (1965).
Protein Synthesis in Rat Liver Chromatin and
Chromatin Fractions
By Doz. C. E. Sekeris, W. Schmid, Dr. D. Gallwitz, and
Dr. I. Lukacs
Physiologisch-Chemisches Institut,
Universitat Marburg (Germany)
In addition to “classical” protein biosynthesis on messenger
RNA and ribosomes, which also occurs in cell nuclei[*],
another type of protein synthesis seems to take place in cell
nuclei. We have found that chromatin isolated from rat liver
cell nuclei can incorporate [14C]leucine into protein. The
new protein-synthesizing system has the foilowing characteristics (cf. Table) :
1. The incorporation of radioactively labelled amino acid
into the protein is resistant to attack by ribonuclease.
2. The protein synthesis is independent of the addition of
amino-acid activating enzymes.
3. The protein synthesis is not stimulated by messenger RNA.
4. The system loses some of its activity on incubation with
The chromatin was isolated from rat liver cell nuclei according to Weiss 141. The preparation (aggregate enzyme 141)
Incorporation of [‘4C]leucine
[countslminx m g of protein]
Chromatin alone [a]
Chromatin ,~ ribonuclease (50 y/ml)
Chromatin i105000 g supernatant
(2 mg of protein)
50 y of messenger R N A
from rat liver
Chromatin i. deoxyribonuclease
la1 Chromatin (Img of protein, preparation obtained after treatment
with deoxycholate, see below), 7.5 (*moles of KCI, 3 vmoles of MgCI2,
80 wnoles of tris-(2-amino-2-hydroxymethyl-l,3-propanediol),
(*mole of ATP, 150 wmoles of GTP, 0.5 (*mole of creatine phosphate,
10 y of creatine phosphokinase, 0.025 (*mole each of 20 amino acids,
and 0.1 wCi of [I4C]leucine with a specific activity of 150 mCi/mmole.
Volume: 0.5 ml. Incubation time: 60 min at 3 7 ° C . The degree of
incorporation was measured as described in [3].
incorporates ribonucleoside triphosphates into R N A ; it
consists of DNA, RNA, and protein in weight ratios of
1 : 2 :10-15. The preparation could be freed from the major
fraction (about 90 %) of its protein by treatment with 0.2 %
sodium deoxycholate in 0.065 M tris buffer at p H 7.9. The
sediment obtained after low-speed centrifugation contained
the whole activity and had D N A : RNA:protein ratios of
1.5. It was suspended in 0.065 M tris buffer at p H 7.9
1 :0.4:
and shaken for 30 min at 37 “C with 100 pg/ml of deoxyribonuclease and 2 p M magnesium chloride. During this treatment, the clear glassy fibrous structure of the preparation disappears and a white powder is obtained. Centrifugation then
separates the suspension into a white precipitate and a cloudy
supernatant. The precipitate contains all of the activity; the
supernatant, which contains liberated deoxynucleotide,
RNA, and protein, inhibits protein synthesis, but the nature
of the inhibitor is not yet known. It is thermally stable and
has a molecular weight of 3000 to 15000. Treatment of the
precipitate with ribonuclease (100 pg/ml) for 20 min at 37 “C
removes proteins and R N A without loss of activity. The
D N A : RNA:protein ratio after this treatment is 2: 1.5:6.5.
Partial digestion with trypsin solubilizes part of the enzyme
in an active state; it occurs in the supernatant after centrifugation.
The protein synthesis is inhibited by histones from rat liver
cell nuclei (up to 90 % inhibition with 1 mg/ml), by protamine
sulfate (up to 98 % inhibition with 1 mgiml), and by antibiotics such as chloromycetin (98 % with 1 mg/ml) and
puromycin (80 % with 0.4mgiml). It was shown by the fingerprint method that leucine is incorporated into peptides. Between 10 and 15 % of the incorporated radioactivity is soluble
in 0.2 N hydrochloric acid. The newly synthesized protein is
not liberated from the enzyme complex. Only a small amount
of the radioactive protein that is not extractable with HCI
dissolves in 8 M urea.
The biological significance of this RNA-independent protein
synthesis is still unknown. Part of the protein formed is
probably structural protein of the chromatin, but the
possibility of a regulatory function must also be considered.
The fact that the protein synthesis on chromatin is influenced
by hormonally active steroids and that it is promoted by
partial hepatectomy support the latter suggestion.
Received: April 29th. 1966
[Z 224 I E ]
German version: Angew. Chem. 78, 601 (1966)
[I] Protein Synthesis in Cell Nuclei, Part 2.
Part 1 : C. E.
Sekeris, W . Schmid, D. Gallwitz, and S. Lucas, Life Sciences 5 ,
969 (1966).
[2] J . H. Freiwter, V . G . Allrey, and A . E. Mirsky, Proc. nat.
Acad. Sci. U.S.A. 46, 432 (1960); T . Wang, Biochirn. biophysica
Acta 49, 108 (1961).
[3] N . Lang and C . E. Sekeris, Hoppe-Seylers Z . physiol. Chem.
339, 238 (1965).
[4] S. B. Weiss, Proc. nat. Acad. Sci. U . S . A . 46, 1020 (1960).
Angew. C h e m . internnt. Edit. / Vol. 5 (1966)
/ No. 6
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