Download On the Nucleotide Sequence of Yeast Tyrosine Transfer RNA

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On the Nucleotide Sequence of Yeast Tyrosine Transfer RNA
J. T. MADISON, G. A. EVERETT,AND H. K. KuNG
U.S. Plant, Soil and Nutrition Laboratory, U.S. Department of Agriculture, Ithaca, New York
Transfer RNA structures presumably get into a
symposium on the genetic code by virtue of the
supposition that there must be three nucleotides
in the tRNA (transfer ribonucleic acid) that
interact with messenger RNA in a specific and
reliable fashion. But since we are looking for only
three nucleotides out of about 80 there is, unfortunately, a lot of nonrelevant material.
Since the methods used to determine the
nucleotide sequence of the tyrosine tRNA from
baker's yeast were the same as those developed
by Holley et al. (1965) during work on the structure
of alanine tRNA, we will not describe them in
detail.
Figure 1 shows the pattern obtained when bulk
yeast soluble RNA is distributed for 220 transfers
in the countercurrent system containing the
additional 2-propanol described by Holley et al.
(1963). In this system the bulk of the RNA has a
very low distribution coefficient and remains in the
low numbered tubes. Tyrosine and phenylalanine
tRNA are moved out and away from most of the
other transfer RNAs.
In 220 transfers tyrosine RNA I and tyrosine
RNA I[ are fairly well separated. The total acceptor activity curve indicates that there is 6 to 8
times as much tyrosine I as tyrosine II RNA.
Both the total RNA and total tyrosine acceptor
activity fall off rapidly to the right of tyrosine
RNA II.
After two redistributions of tyrosine RNA I,
the pattern in Fig. 2 was obtained. The total
tyrosine acceptor activity and ultraviolet absorbance curves coincide fairly well. The tyrosine
acceptor specific activity decreasing on the left
side of the peak probably means that there is
another RNA that distributes just behind the
tyrosine tRNA. The best indication of the purity
of the tyrosine I:r
comes from analyses of
nuclease digests of this material. All the evidence
is consistent with the view that the tyrosine RNA
in fractions 100 to 130 was about 80% pure. But
this material has proved to be adequate for sequence studies.
The fragments obtained by pancreatic RNase
digestion of the tyrosine I~NA are shown in Table I.
The different size fragments were separated by
chromatography on DEAE cellulose in 7 M urea,
0.02 M Tris, pH 7 (Tomlinson and Tener, 1962).
The nucleosides were not extensively studied;
the mononucleotides were desalted and identified
after two-dimensional paper chromatography. The
di- through pentanucleotides were separated by
chromatography on DEAE cellulose in 7 M urea,
0.1 M formic acid (Rushizky and Sober, 1964), and
sequences assigned. The sequence of the one heptanucleotide was determined by partial snake venom
phosphodiesterase digestion (Holley, Madison, and
Zamir, 1964) and treatment with micrococcal
nuclease (Sulkowski and Laskowski, 1962). The
R
01
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of yeast bulk sRNA.
i
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,
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120
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160
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410
MADISON, E V E R E T T , AND KUNG
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NO.
pCp is the first nucleotide other than pGp to be
found at the 5' phosphate terminus of a purified
tRNA, although all four major diphosphonucleosides have been found in alkaline hydrolysates
of bulk sRNA (Bell et al., 1964). The nucleotide
abbreviated MeA- has not been conclusively
identified, but it is probably a derivative of
Nl-methyladenylic acid.
The products of RNase T1 digestion are shown
in Table 2. Chromatography on a DEAE cellulose
column, 8 ft by 0.3 cm, in 7 M urea, with a sodium
acetate gradient, pH 6, was sufficient to separate
TABLE 1. OLIGONUCLEOTIDES OBTAINED FROM TYROSINE
R N A I BY DIGESTION WITH PANCI~EATIC RIBONUCLEASE
COH
9-11 C5-6 U 2 DiHUMeCVpCG-TG-CDiMeG-C-
G-MeA-CA-2MeG-CG-G-U21 O M e G - G - D i H U A - D i M e A - A - VA-A-G-DiHUG-G-G-CA-A-G-A-CA-A-G-G-CG-A-G-A-DiHU-
G - V-
G-G-G-A-G-A-C-
most of the oligonucleotides shown. Paper electrophoresis, chromatography on DEAE cellulose in
7 M urea, 0.1 M formic acid or chromatography on
DEAE Sephadex, eluting with ammonium carbonate, were used when necessary.
Dihydrouridylic acid (DiHU-) is concentrated
in the oligonucleotides DiHU-DiHU-2' O MeGG- and D i H U - D i H U - D i H U - A - A - G - and is also
found in the tetranucleotide, A-DiHU-hMeC-G-.
The hexanucleotide C-C-C-C-C-G- contains the
140
IEIO
FIGURE 2. R e d i s t r i b u t i o n of y e a s t
tyrosine R N A I.
longest run of a single nucleotide that has yet been
found in an RNA. The 3' hydroxyl terminus is
A-C-Coil, indicating that, as in the case of the
yeast alanine tRNA, the terminal adenylic acid
residue is missing.
Digestion of the intact RNA and digestion of the
5' phosphate half of the molecule with RNase T1
at 0 ~ produced large fragments. These fragments,
when separated and digested with nucleases and
the products analyzed, gave enough information
to specify a unique sequence (Madison, Everett,
and Kung, 1966). The nucleotide sequence of the
tyrosinc RNA is shown in Table 3, along with that
of the alanine tRNA. Yeast alanine tRNA has
77 nucleotides, while the tyrosine RNA has 78
nucleotides. The extra nucleotidc in tyrosine RNA
has been placed beside the DiHU in the next to
bottom line. The reason for placing it there will
be more apparent shortly.
The solid line between the sequences indicates
that the same nucleotide is present in the same
position in both RNAs. Except for the G - T - ~ - C G - and A-C-C-AoH sequences which have been
known to be common to many tRNAs (Zamir,
Holley, and Marquisee, 1965; Smith and Herbert,
T A B L E 2. O L I G O N U C L E O T I D E S O B T A I N E D FROM T Y R O S I N E R N A
I
BY DIGESTION WITH RIBONUCLEASE T1
8 GA-C-U-GC-DiMeGp!
C-A-A-GA-C-COH
A-DiHU-hMeC-GC-GMeA-C-U-C-G3 A-GC-C-A-A-GU-A-2MeGC-C-C-C-C-GDiHU-DiHU-2' O MeG-GDiHU-DiHU-DiHU-A-A-GT~-C-GpC-U-C-U-C-G~#-A-DiMeA-A-v-C-U-U-G-
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YEAST TYROSINE tRNA
411
TABLE 3. A COMPARISOI~ OF THE NUCLEOTIDE SEQUEI~CES OF YEAST ALANINE AND TYROSllVE t R N A s
20
2Me
D i l l D i l l Me D i l l D i l l D i l l
DiMe
pC-U-C-U-C-G-G-U-A-G-C--C-A-A-G-U
- U -G-G-U - U - U-A-A-G-G-C-G-C-A-A-G-A-C-U-G-~-
Tyr
1Me
pG-G-G-C-G-U-G-U-G-G-C-G-C-G-U-A
Ala
Dill
Dill
DiMe
- G- U-C-G - G - U-A-G-C-G-C-G-C-U-C-C-C--U-U-I-G-
DiMe
D i l l 5Me
Me
A- A-A-~0-C-U-U-G-A-G-A-U - C-G-G-G-C-G-T-~o-C-G-A-C-U-C-G-C-C-C-C-C-G-G-G-A-G-A-C-C-AOH
T y r (cont.)
Me
*
C- I-~a-G-G-G-A-G-A-G - U - C-U-C-C-G-G-T-y~-C-G-A-U-U-C-C-G-G-A-C--U-C-G-U-C-C-A-C-C-AOH
1965), there is only one place where the same four
nucleotides occupy identical positions in the two
RNAs: G-C-DiMeG-C-. I f it were not for the
additional A- in tyrosine RNA, there would be
five nucleotides in identical positions in the bottom
two rows.
Figure 3 shows, however, that it is possible to
construct very similar base-paired structures in
spite of the limited similarities in sequences. This
model was first suggested by J. R. Penswick and
was further refined by E. B. Keller. Only 11 out of
the 31 nucleotides shown in Table 3 as being
in the same location in both RNAs are located in
the regions shown here as being double stranded, so
that the similar base-paired structures are not the
result of those nucleotides that are in the same
position in both chains.
In each case there is an upper "stem" of seven
base pairs. In both cases there is one G : U pair
(Crick, 1966) in this "stem". The alanine RNA
has two Us opposite each other that presumably
can not form a base pair. Both of the right-hand
and lower limbs consist of five Watson-Crick base
pairs terminated by a seven residue loop. The
left-hand loop of the tyrosine RNA has three
G:C type base pairs and a twelve residue loop.
The alanine RNA has the same number of nucleotides in the left-hand limb, but in this case four G: C
base pairs are possible, leaving a ten residue loop.
The transitions between the double-stranded
regions are also very similar. The only difference
is in the transition from the right-hand to the
lower limb where the tyrosine RNA has five
unpaired nucleotides, while the alanine RNA has
four nonbase-paired residues. This difference accounts for the difference in the number of nucleotides in the two molecules.
The anticodons are almost certainly the G - ~ - A
and I-G-C sequences in the lower loops. The
codons for tyrosine, U - A - U and U-A-C (Nirenberg
et al., 1965) would be expected to bind with
A-U-A or G - U - A (or possibly with A - U - G if the
tRNA and mRNA chains were parallel). There are
Ala
(cont.)
no A - U - A or A - U - G sequences in the tyrosine
RNA. The only G - U - A sequence is at the junction
of left-hand and upper limbs. The sequence in the
alanine RNA that corresponds to the G-U-A is
G-U-1MeG, an unlikely candidate for the alanine
anticodon.
The other sequence considered as an alanine
anticodon, the C-G-G sequence between the
DiHUs in the left-hand loop (Holley et al., 1965),
seems unlikely since the region between the
DiHUs in the tyrosine RNA does not look like an
anticodon.
Consideration of inosine (I) containing sequences
in serine (Zachau, Dutting, and Feldmann, 1966
and in this volume) and valine tRNAs (Armstrong
et al., 1964) also strongly suggests that I is part of
the anticodon.
It is interesting that the presumed anticodons
are flanked in both cases by U on one side and a
modified A on the other.
Trinucleotide binding data (Doctor, Loebel, and
Kellogg, this volume) indicate that this tyrosine
RNA will react with both U - A - U and U-A-C,
showing that G in the wobble position of Crick
(1966) will pair with both U and C. Similar experiments with alanine tRNA have not led to such
clear-cut results. Both Ledcr and Nirenberg, and
Keller and Ferger (unpubl.) have found that
purified yeast alanine RNA will bind with G-C-A,
G-C-C, and G-C-U, but that it will bind only
poorly, if at all, with G-C-G. So it appears that I
in the wobble position will tolerate A, C, and U,
but its reaction with G is still in doubt.
From a simple-minded point of view, there does
not seem to be an obvious reason why the anticodon
for tyrosine should contain pseudouridine instead
of U. Pseudouridine has an additional N - H
available for hydrogen bonding. This might be a
reason for its presence.
Consideration of the locations of modified bases
suggests that there are enzymes that act on bases
in certain locations. That is, the specificity of the
modifying enzymes may be based not on the
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412
MADISON, EVERETT, AND KUNG
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YEAST TYROSINE tRNA
nucleotide sequence in which the modified nucleotide occurs, but rather on its location in the three
dimensional structure of the molecule. Since ~ is
found only in the lower and right-hand loops,
there may be an enzyme that converts U to
in the lower loop and another enzyme that does
the right-hand loop (or the same enzyme might
work on both loops). In either case the ~ in the
tyrosine anticodon might be an incidental product
of an enzyme whose real purpose is to change U
to v2 in the wobble position (Crick, 1966).
The basis for suggesting that the modifying
enzymes act on nucleotides in certain parts of
the molecule is more apparent in the case of the
DiHUs. The fact that D i H U - is found only in the
left-hand loop and in the lump between the righthand and lower limbs suggests there may be an
enzyme (or enzymes) whose specificity is to
hydrogenate U when it is in these locations. The U
in alanine RNA that is only approximately onehalf converted to DiHU (U*) might be explained
by its being less accessible than the DiHU in the
comparable position in tyrosine RNA, since the
alanine RNA lump has one less nucleotide. On
this basis, in order to explain the unhydrogenated
U in the alanine RNA left-hand loop, we have to
propose that the enzyme can only reach Us that
have at least two nucleotides between them and
the double-stranded region. The same limitation
would have to be placed on the ~ forming enzyme.
The locations of the other modified bases can be
explained fairly well on the basis of accessibility.
Alanine RNA does not contain any modified base
in a position shown here as being double-stranded.
The tyrosine RNA, however, has a ~ next to the
lower loop and a 2MeG in the left-hand limb.
Both of these bases presumably can form base
pairs. Perhaps these double-stranded regions can
open up enough to allow an enzyme to modify
bases in these positions.
Those bases that would be expected to be unable
to form base pairs--namely 1 MeG, N2-DiMeG,
1 MeI, Ne-DiMeA,--are all shown here as being in
single-stranded regions. It seems possible that
their function is to prevent the formation of basepaired double helices in the regions where they
are located, and perhaps to assure that base
pairing does take place between the correct pairs of
nucleotides.
In 0.02 M MgCl~ (Penswick and Holley, 1965)
RNase T1 hydrolyzes only the phosphodiester bond
next to the G in the lower loop in both tRNAs.
This is reasonable, since it indicates that under
these conditions the anticodon is exposed. But, in
order to explain this kind of specificity, it must be
supposed that the Gs in the other loops are
protected from attack by the enzyme. It could be
413
that in 0.02 M MgC12 the two lateral arms are
folded together, perhaps even with the formation
of base pairs, leaving the bottom loop exposed.
Other types of folding undoubtedly occur also.
We wish we could identify the activating-enzyme
recognition site. At first glance, it would seem that
the enzyme recognition site should be one of the
single-stranded regions. Since the right-hand loops
of different tRNAs are so similar and the lower
loop has the task of presenting the anticodon in
correct conformation, these would seem unlikely
areas. The variation in the ability of activating
enzymes from one species to charge the tRNA from
another species argues against the anticodon
being the activating-enzyme recognition site.
Results obtained by Penswick (1966) also indicate
that the anticodon is not likely to be part of the
enzyme recognition site; these experiments show
that the alanine tRNA retains its acceptor activity
after it has been cleaved by ribonuclease T1 at the
G of the anticodon. The cleaved RNA retains its
acceptor activity only as long as the two halves
remain bonded together. Earlier experiments of
Nishimura and Novelli (1965) also lead to the same
conclusion.
It is not altogether coincidental, therefore, that
the regions left for the activating enzyme to
recognize are those that show some variation
between the two RNAs. The left-hand loop and
the "lump" between the right-hand and lower
limbs show variations in both size and nucleotide
composition, either of which an enzyme could
probably use to recognize which tRNA it was
about to aminoacylate.
The G :U base pair in the upper stem might be
important. Is it possible that the activating
enzymes could extract enough information from
the G :U pair and other features of this doublestranded region for it to act as a recognition site ?
So much for speculation on what the tRNAs look
like.
When tyrosine RNA II was redistributed for
1170 transfers, the pattern in Fig. 4 was obtained.
The ultraviolet absorbance and tyrosine acceptor
activity curves generally follow each other. The
only known contaminant, phenylalanine tRNA,
is at a minimum where the tyrosine acceptor
activity is the highest.
Digestion of the fraction having the highest
tyrosine acceptor activity with RNase T1 and
chromatographing it on an 8 ft by 0.3 cm column
of DEAE cellulose in 7 ~ urea gives the pattern
shown in Fig. 5. The pattern obtained from tyrosine
RNA I is included for a comparison. The only
obvious difference between the two is that the
A-C-Con in tyrosine I is replaced by A-C-C-Ao~
in tyrosine RNA II. Otherwise tyrosine II has every
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Press
414
MADISON, EVERETT, AND KUNG
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In every case it seems likely that the fragments
detected in tyrosine I are also present in tyrosine
II, with considerable impurities in some cases.
The C-C-C-C-C-G- in tyrosine II, for example, is
contaminated with appreciable amounts of U-, A-,
and a MEG-. The three tetranucleotides that
chromatograph together could not be identified
conclusively in tyrosine R N A I I . DiHU was
detected, but not 5 MeC-. The ratio of U to C is
not what would be expected. But everything
considered, the similarities between the two RNAs
are so great that it seemed likely that the only
difference was that tyrosine RNA II contained
the terminal pA residue that is absent from tyrosine I. From the standpoint of the genetic code,
the only really interesting difference between the
two would have been for tyrosine RNA II to have
A-U-A as the anticodon. Since, however, ~p-ADiMeA-A-~p-C-U-U-G- was produced by RNase
T1 digestion from tyrosine RNA II, the anticodon
in tyrosine II is also probably G-~-A. Lacking the
stamina to pin down each peak unequivocally, we
did the following experiment. Eight mg of tyrosine
RNA I was incubated with C14-ATP and a crude
-~ ,,
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120
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160
FRACTION
NO.
I
I0
180
FIGURE 4. R e d i s t r i b u t i o n of y e a s t t y r o s i n e R N A I I .
peak that tyrosine I does, plus a couple of extras
that might be impurities.
Table 4 shows the base compositions of the peaks
shown in Fig. 5 along with the structures assigned
to the corresponding fragments from tyrosine RNA I.
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FIGURE 5. C h r o m a t o g r a p h y
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o f t h e p r o d u c t s o f r i b o n u c l e a s e T1 d i g e s t i o n o f y e a s t t y r o s i n e R N A s I a n d I I .
Downloaded from symposium.cshlp.org on September 15, 2016 - Published by Cold Spring Harbor Laboratory
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YEAST TYROSINE tRNA
415
T A B L E 4. N U C L E O T I D E COMPOSITION OF THE PRODUCTS OF RIBOI~UCLEASE T 1 D I G E S T I O N
oF TYROSXNERNA II
Structure assigned to
fragment from Tyr RNA I
Nucleotide composition of corresponding
peak from Tyr RNA II
AG--c-C~ 1
C-DiMeGp!J
C-G2 A-G-
GC-1.00, DiMeG-1.35
C-0.93, G-1.00, U-0.22
A-4.51, G-2.00
C-1.84, Aoa 0.61
U-1.72, A-1.30, 2MEG-1.00, G-0.59
MeA-1.21, C-2.56, U-1.63, G-1.00
DiHU-1.94, 2'OMeG-G-1.00
T-l.17, ~f-l.07, C-1.28, G-1.00
DiHU-present, A-1.74, G-1.00
C-0.72, A-1.44, U-1.28, G-1.00
I
U-A-2MeGMeA-C-U-C-GDiHU-diHU-2'OMeG-GT-yJ-C-GA-DiHU-5MeC-G--]
A-C-U-G|
C-A-A-G_]
C-C-A-A-GDiHU-DiHU-DiHU-A-A-GC-C-C-C-C-GpC-U-C-U-C-Gvj-A-DiMeA-A-~-C-U-U-G-
I
c-2.92, A-2.24, G-1.00
DiHU-2.91, A-2.38, G-1.00
C-6.7, G-0.74, U-2.20, A-I.10, MEG-1.00
pC-1.20, U-1.60, C-1.58, G-1.00, A-0.26
~-2.34, A-1.96, DiMeA-1.04, C-0.75, U-2.52, G-1.00
enzyme preparation from yeast containing the
terminal-adding enzyme. The incubation mixture
included 1 ml 0.5 M glycine, p H 9.5; 1 ml 0.05
phosphoenolpyruvate, 0.2 ml 0.5 M MgC12, 0.1 ml
0.02 M ATP, 1.5 ml Cta-ATP (1 #c/ml, 2 #c/#mole)
and 1 ml of the crude enzyme preparation. After
12 min at 37 ~ the mixture was extracted three
times with phenol and three times with ether, and
the RNA was separated from the Ct4-ATP by gel
filtration on Sephadex G-25. The material excluded
by the gel contained about 4500 count/min. These
fractions were pooled and added to 6 g of crude
yeast sRNA and distributed b y countercurrent
distribution for 200 transfers. The pattern shown
in Fig. 6 was obtained. The vast majority of the
counts distributed with tyrosine II, showing that
it is possible to change the countercurrent behavior
of tyrosine RNA I by the addition of the terminal
pA. Since tyrosine I I contains the terminal pA, it is
unnecessary to postulate any other difference
between tyrosine RNA I and tyrosine RNA II.
Similar results with yeast phenylalanine t R N A
have been reported by RajBhandary et al. in this
volume.
For those who would like a second species of
tyrosine t R N A for control purposes or for any other
reason, we can only nominate the fraction to the
right of tyrosine II. While it m a y not be significant,
we have seen it more than once. There is, however,
very little RNA or tyrosine acceptor activity in
this region. There is, at most, probably only onethousandth as hluch of this material as there is
tyrosine RNA I plus II, precluding any effort, on
our part, to look at its structure.
In summary: the nucleotide sequence of yeast
tyrosine transfer RNA strongly supports the
12
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~
140
.:
o... ~ ..."
t
FIGURE 6. Countercurrent distribution
of yeast tyrosine tRNA containing terminal C14-adenylic acid.
3o~
~
,
8 0L
100
F R A C T I O N NO.
"..
u
u
20"
....~1/
,
120
:
160
~o~
Downloaded from symposium.cshlp.org on September 15, 2016 - Published by Cold Spring Harbor Laboratory
Press
MADISON, E V E R E T T , A N D K U N G
416
Penswick-Keller cloverleaf model for transfer RNA.
The anticodon for tyrosine RNA I and tyrosine
RNA II is probably G-v-A. The removal of the
3' hydroxyl terminal adenylic acid from tyrosine
tRNA significantly affects its behavior upon
countercurrent distribution.
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BELL, D., R. V. TOMLINSON, and G. M. TENER. 1964.
Chemical studies on mixed soluble ribonucleie acids
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CRICK, F. H. C. 1966. Codon, anticodon relationships: The
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HOLLEY, R. W., J. APGAR, G. A. EVERETT, J. T. MADISON,
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HOLLEY, R. W., J. T. MADISON, and A. ZAMXR. 1964. A
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MADISON, J. T., G. A. EVERETT, and H. KUNG. 1966.
Nucleotide sequence of a yeast tyrosine transfer RNA.
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NISHIMURA, S., and G. D. NOVELLL 1965. Dissociation of
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PENSWICK, J. R. 1966. Contributions to the structure of
alanine transfer rihonucleie acid from Saccharomyces
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PENSWICK, J. R., and R. W. HOLLEY. 1965. Specific cleavage of the yeast alanine RNA into two large fragments.
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RUSHIZKY, G. W., and H. A. SOBER. 1964. Chromatography of tri- and tetranucleotides from pancreatic
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SMITH, C. J., and E. HERBERT. 1965. Terminal nucleotide
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SULKOWSKI, E., and M. LASKOWSKI, Sr. 1962. Mechanism
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DISCUSSION
C. McLAuoHLIN: Where are the cleavage points
produced during partial enzymatic digestion located
on your models for tyrosine and alanine tRNA ?
One suspects that the initial sites of attack should
occur in single stranded regions.
J. T. MADISON: In 0.02 M MgC12 the initial site of
attack by RNase T1 is at the G in the lower loop
(Fig. 3). In the absence of Mg, I don't know what
the initial sites of attack are. The fragments
produced by partial digestion by RNase T1 that
were utilized in the reconstruction of the nucleotide
sequence of tyrosine tRNA were the result of
cleavage at 4 locations in double-stranded regions
and 4 in single-stranded regions of the model
(Fig. 3). In the case of the alanine tRNA, 5 out
of 9 of the principal points of cleavage were in
single-stranded regions. In both cases, however,
the fragments produced by the cleavages described
were isolated after digestion for 1 hour with RNase
T1. I don't think that it is safe to assume that
fragments isolated after this length of time are
the products of the initial attack by the enzyme.
Downloaded from symposium.cshlp.org on September 15, 2016 - Published by Cold Spring Harbor Laboratory
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On the Nucleotide Sequence of Yeast Tyrosine Transfer
RNA
J. T. Madison, G. A. Everett and H. K. Kung
Cold Spring Harb Symp Quant Biol 1966 31: 409-416
Access the most recent version at doi:10.1101/SQB.1966.031.01.053
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