Download Early days of tRNA research: Discovery, function, purification and

Early days of tRNA research: Discovery, function, purification and sequence analysis
439
Early days of tRNA research: Discovery, function, purification and
sequence analysis
UTTAM L RAJBHANDARY* and CAROLINE KÖHRER
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
*Corresponding author (Fax, 617-252-1556; Email, [email protected])
Research on tRNA and aminoacyl-tRNA synthetases can
be traced back to the mid-fifties when Mahlon Hoagland
in Paul Zamecnik’s group (figure 1) working on the
development of a cell free protein synthesis system from rat
liver discovered an enzyme in the pH 5-insoluble fraction
(pH 5 enzyme) which activated amino acids in the presence
of ATP to yield aminoacyl-adenylates (Hoagland et al 1956;
Zamecnik 2005). The formation of aminoacyl-adenylates
was demonstrated using amino acid-dependent ATP-PPi
exchange and by their reaction with neutral hydroxylamine
to form amino acid hydroxamates1 (figure 2). The Zamecnik
laboratory then went on to show that an enzyme in the same
pH 5 fraction transferred radio labelled amino acid (14Cleucine) to an RNA acceptor2 (figure 3) and that the 14Cleucine attached to the RNA was subsequently transferred
to protein in a protein synthesizing system (Hoagland et al
1957, 1958), indicating an important role for this RNA as an
intermediate carrier of the amino acid in protein synthesis
(table 1). Independently, Ogata and Nohara (1957) obtained
evidence for transfer of amino acid by the pH 5 enzyme
fraction to an RNA. At the same time, Holley (1957)
showed that the pH 5 enzyme catalyzed alanine-dependent
ATP-AMP exchange that was sensitive to ribonuclease.
This result also supported the notion of amino acid transfer
to RNA although the ribonuclease sensitive ATP-AMP
exchange was observed only in the presence of alanine and
not other amino acids. Zamecnik and his colleagues named
the RNA in the pH 5 enzyme fraction soluble RNA3 (sRNA).
This RNA is now known as tRNA. It was soon demonstrated
that the amino acid-activating enzyme in the pH 5 fraction
also catalyzed the transfer of the activated amino acid to the
tRNA (Berg and Ofengand 1958; Lipmann et al 1959). This
enzyme is now known as aminoacyl-tRNA synthetase.
The discovery of tRNAs and aminoacyl-tRNA
synthetases stimulated much interest in these classes of
molecules. The groups of Berg (figure 4), Lipmann, Novelli,
Schweet and Zamecnik were among the early contributors
(Berg 1956, 2003; Berg et al 1961; Davie et al 1956;
DeMoss and Novelli 1956; Schweet et al 1958; Zamecnik
et al 1958). It was quickly found that each aminoacyltRNA synthetase is specific for an amino acid and that each
enzyme attaches a specific amino acid onto a cognate tRNA.
Work from several laboratories including those of Potter,
Canellakis, Herbert and Zamecnik led to the conclusion that
tRNAs contained a common sequence, -CCA, at the 3’-end
(Canellakis 1957; Hecht et al 1958a; Heidelberger et al
1956), that is necessary for transfer of the amino acid to the
tRNA (Hecht et al 1958b). A question of much interest early
1
3
1.
Introduction
This review is based on a talk presented at the 21st
International tRNA Conference held at the Indian Institute
of Science in Bangalore, India in December 2005. It covers
the early days of tRNA research (from 1955–1980) and
includes a brief description of the discovery of tRNA, its
function in protein synthesis and methods for its purification
and sequence analysis.
2.
Discovery of tRNAs and aminoacyl-tRNA
synthetases
Further confirmation of this came later from conversion of
synthetic aminoacyl-adenylates to ATP in the presence of
pyrophosphate and enzyme (Berg 1958; DeMoss et al 1956).
2
Interestingly, as pointed out by Hoagland, the discovery of 14Cleucine transfer to an RNA acceptor was the result of a control
experiment (Hoagland 1996).
http://www.ias.ac.in/jbiosci
Called soluble RNA because most of it was found in the
supernatant fraction of a 105,000 g centrifugation step and not
in the sedimentable microsomal fraction. When the supernatant
fraction is adjusted to pH 5, the sRNA precipitates along with
the aminoacyl-tRNA synthetase, pH 5 being the approximate
isoelectric point of tRNA-aminoacyl-tRNA synthetase complexes.
J. Biosci. 31(4), October 2006, 439–451, © Indian
of Sciences
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J.Academy
Biosci. 31(4),
October 2006
Uttam L RajBhandary and Caroline Köhrer
440
Figure 3. A soluble ribonucleic intermediate in protein synthesis.
14
C-labelling of cellular RNA fractions from rat liver (from:
Hoagland et al 1958).
Figure 1. Paul C Zamecnik (courtesy of P C Zamecnik).
Rat Liver Homogenate
105,000 x g
Supernatant
pH 5.2
Precipitate
(sRNA and synthetases)
The pH5.2 precipitate
(i)
Activates amino acids in the presence
of ATP to form aa~AMP as assayed
by amino acid-dependent ATP:PPi
exchange and by formation of amino
acid hydroxamates
(ii)
Incorporates radiolabeled amino acids
into sRNA
Figure 2. Discovery of synthetases and sRNA.
J. Biosci. 31(4), October 2006
on was where and how the amino acid was attached to the
tRNA. Zachau’s work in Lipmann’s laboratory established
that amino acids were attached to the 3’-terminal A residue
of the tRNA through aminoacyl-ester linkages to the 2’- or
the 3’- hydroxyl groups of ribose (Zachau et al 1958). Thus,
the emerging view at the end of the fifties was that amino
acids are first activated by aminoacyl-tRNA synthetases to
form aminoacyl-adenylates, the activated amino acids are
then transferred to tRNA acceptors and finally the amino
acids attached to the tRNAs are transferred to protein. The
tRNAs turned out to be much larger than the small molecules
proposed by Crick to function as adaptors, in his well known
albeit unpublished “Adaptor Hypothesis” designed to
explain how DNA sequences might be translated to amino
acid sequences in proteins.
3.
Sequences of tRNAs
Emphasis in the sixties switched to work on the genetic
code and determination of primary sequence of tRNAs. As
adapter molecules, which translate the nucleotide sequence
in mRNAs to amino acid sequences in proteins, tRNAs
played a central role in the elucidation of the genetic code
and in the advent of the early days of molecular biology.
The first tRNA sequenced was that of yeast alanine
tRNA by Holley (figure 5) and coworkers in 1965 (Holley
et al 1965). Three possible secondary structures. were
proposed for this RNA, one of which is the now well
known cloverleaf structure (figure 6). Sequences of several
Early days of tRNA research: Discovery, function, purification and sequence analysis
441
Table 1. Transfer of 14C-leucine from pre-labelled pH 5 enzyme fraction to microsome protein.
GTP
PEP/pyruvate kinase
RNA*
Protein*
Before incubation
+
+
478
22
After incubation
+
+
182
433
After incubation
–
+
116
67
After incubation
+
–
62
101
After incubation
–
–
29
23
*Total cpm in RNA and protein. Microsome suspension and pH 5 enzyme fraction pre-labelled with
14
C-leucine were incubated with GTP, PEP, and pyruvate kinase as indicated (adapted from: Hoagland
et al 1958).
Figure 5. Robert W Holley.
Figure 4. Paul Berg.
tRNAs followed in quick succession (1966–1967), those of
two closely related serine tRNAs by Zachau (figure 7) and
coworkers (Zachau et al 1966), tyrosine tRNA by Madison
and coworkers (Madison et al 1966) and phenylalanine
tRNA by RajBhandary, Khorana (figure 8) and coworkers
(RajBhandary et al 1966, 1967). The availability of several
tRNA sequences and the finding that they could all be folded
into a cloverleaf structure established the cloverleaf as the
common secondary structure of tRNA. It also led to the
identification of anticodon sequences of tRNAs. Knowledge
of the anticodon sequences of several tRNAs along with
the degenerate and systematic nature of the then newly
established genetic code led Crick to propose in 1966 a set of
base-pairing rules for codon-anticodon interactions in “The
Wobble Hypothesis” (Crick 1966). This hypothesis was
confirmed soon after by experiments showing that purified
yeast phenylalanine tRNA with the anticodon sequence
GmAA could read either of the phenylalanine codons UUU
and UUC in an in vitro protein synthesis system (Söll and
RajBhandary 1967).
The first few tRNAs sequenced were all from yeast.
Sequence analysis required large amounts of purified tRNAs
(usually in the order of several hundred milligrams to a
gram) and baker’s and brewer’s yeast provided a ready and
inexpensive source of total tRNAs that could be used for
purification.
4.
Methods for sequence analysis of tRNAs
The strategy used for sequence analysis of tRNAs above
is now classical and consisted of the following steps:
(i) complete cleavage of the purified tRNA with basespecific nucleases such as pancreatic RNase (pyrimidine
specific) or T1-RNase (G-specific) followed by separation
of the oligonucleotide fragments from each other and
establishment of their sequences; (ii) isolation of longer
fragments of the tRNA by limited (partial) digestion with
J. Biosci. 31(4), October 2006
442
Uttam L RajBhandary and Caroline Köhrer
Figure 8. H Gobind Khorana.
Figure 6. Structure of a ribonucleic acid. Schematic representation
of three conformations of tRNAAla (reprinted from Holley et al
Science, 147, pp 1462–1465. “Structure of a ribonucleic acid”.
Copyright 1965, with permission from Science).
Figure 7. Hans G Zachau.
J. Biosci. 31(4), October 2006
pancreatic or T1-RNase and analysis of their sequences
to obtain overlaps between the oligonucleotide fragments
present in a complete digest; and finally (iii) assembly of the
fragments into a unique sequence.
Sequence analysis of oligonucleotide fragments present
in digests of tRNA requires that they be separated from each
other. The use of DEAE-cellulose ion-exchange columns
in the presence of urea (7 M) as a denaturant described
by Tener’s group (Tomlinson and Tener 1963) allowed
separation of the oligonucleotides on the basis of their
charge (size) and to some extent the purine/pyrimidine
ratio. It was a critical and a most timely development and
was used by all the laboratories working on tRNA sequence
analyses. Below, we provide some examples for separation
of fragments of yeast phenylalanine tRNA and establishment
of the sequence of this tRNA.
Figure 9 shows the separation of oligonucleotide fragments
present in complete T1-RNase digests of yeast phenylalanine
tRNA and the quantitation and sequence of the nucleotides
and oligonucleotides in the individual peaks. Virtually every
one of the oligonucleotides are separated from one another
and their sequences could be established by further digestion
to mono-, di- or tri-nucleotides with other enzymes, followed
by paper chromatographic separation of the products
and their identification by spectrophotometric analysis
(RajBhandary et al 1968). Eventually, every oligonucleotide
is cleaved to mononucleotides and the mononucleotides are
identified. The use of spectrophotometry for identification of
the mononucleotides allowed characterization of the many
modified nucleotides that are found in tRNAs.
Analysis of a complete digest of the same tRNA with
pancreatic RNase yields a different set of fragments and the
overlap of sequences between the T1-RNase and pancreatic
Early days of tRNA research: Discovery, function, purification and sequence analysis
443
(A)
(B)
Figure 9. Yeast tRNAPhe. Products obtained by digestion with T1-RNase. Column: DEAE-cellulose (1 x 103 cm). Gradient: 0.02 M TrisHCl pH 7.5, 7 M urea from 0 to 0.3 M NaCl (from: RajBhandary et al 1968).
Figure 10. Yeast tRNAPhe. Products of partial digestion with T1-RNase. Column: DEAE-cellulose (0.7 x 200 cm). Gradient: 0.02 M TrisHCl pH 7.3, 7 M urea from 0 to 0.5 M NaCl (from: RajBhandary and Chang 1968).
J. Biosci. 31(4), October 2006
444
Uttam L RajBhandary and Caroline Köhrer
(A)
(B)
Figure 11. Yeast tRNAPhe. (A) Elution pattern obtained upon re-chromatography of peak 24 of figure 8 at acidic pH. (B) Chromatographic
pattern obtained after T1-RNase digestion of peak 24a from (A) (from: RajBhandary and Chang 1968).
Figure 12. Yeast tRNAPhe. Products of partial digestion and derivation of the total sequence (from: RajBhandary and Chang 1968).
RNase fragments yields sequence information of longer
oligonucleotides. This information is, however, not enough
to derive a unique sequence for the tRNA. It is, therefore,
necessary to isolate longer fragments produced by limited
digestion of the tRNA with T1-RNase or pancreatic RNase,
carried out for short times, at reduced temperature and in
the presence of Mg2+ to retain the tertiary structure of the
tRNA (Litt and Ingram 1964; Nishimura and Novelli 1963).
Under such conditions, the tRNA is cleaved to yield longer
oligonucleotide fragments (RajBhandary and Chang 1968),
which can be separated on long columns of DEAE-cellulose
(figure 10, peaks 19–27). These longer oligonucleotides can
be further separated from each other by chromatography on
J. Biosci. 31(4), October 2006
7 M urea-DEAE-cellulose columns at acidic pH (pH 3.5) and
the longer oligonucleotides in the individual peaks subjected
to complete digestion with T1-RNase for identification of
the component oligonucleotides (figure 11). The overlap of
sequences between these longer oligonucleotide fragments
is then used to derive a unique sequence for the full-length
tRNA (figure 12).
5.
Sequence analysis of uniformly 32P-labelled tRNAs
An important next step in sequence analysis of tRNAs
was the development by Sanger (figure 13) and co
workers of methods for separation of uniformly 32P-labelled
Early days of tRNA research: Discovery, function, purification and sequence analysis
445
Figure 13. Fred Sanger.
oligonucleotides by two-dimensional electrophoresis (figure
14, Anderson and Smith 1972) and a rapid method for their
sequence analysis based on changes in electrophoretic
mobility brought about by successive removal of
nucleotides from the 5’-end with spleen phosphodiesterase,
a 5’-exonuclease (Sanger et al 1965). It led to establishment
of sequences of several Escherichia coli tRNAs and other
RNAs (for example ribosomal RNAs and viral RNAs) that
could be labelled in vivo with 32P and purified readily. One of
the first important results to come out of this work was that
a tyrosine inserting amber suppressor tRNA was produced
by mutation of a single nucleotide in the anticodon sequence
of the tRNA (Goodman et al 1968). This approach also
provided the first sequence of an initiator tRNA, that is used
exclusively for the initiation step of protein synthesis (Dube
et al 1968).
6.
New methods for purification of tRNAs
The yeast tRNAs used for sequence analysis above
were all purified using the principle of countercurrent
distribution pioneered by Lyman Craig (Craig 1952).
While this technique was most useful for early studies
of tRNAs, it was somewhat unwieldy and separation of
tRNAs, while effective, was quite time consuming. With
the sequences of several tRNAs established, the emphasis
now was on biophysical and structure-function studies of
tRNAs, requiring purified tRNAs in large amounts from
Figure 14. Fingerprint of in vivo 32P-labelled tRNA. T1-RNase
products from su+ tRNA. First dimension: electrophoresis on
cellulose acetate paper in pyridine acetate pH 3.5, 7 M urea. Second
dimension: electrophoresis on DEAE-cellulose paper in 7% formic
acid (reprinted from Anderson and Smith, J. Mol. Biol., 69, pp
349–356. “Still more mutant tyrosine transfer ribonucleic acids”.
Copyright 1972, with permission from Elsevier).
a variety of sources. Fortunately, about this time, several
groups described methods for tRNA purification including
the use of benzoylated-DEAE-cellulose (BD-cellulose)
by Tener and coworkers (Gillam et al 1967), DEAESephadex by Nishimura and coworkers (Nishimura et al
1967), reversed-phase chromatography (RPC) by Kelmers,
Novelli and coworkers (Pearson et al 1971) and Sepharose
4B by Holmes, Reid, Hatfield and coworkers (Holmes et al
1975).
The BD-cellulose column separates tRNAs based on
interaction of exposed hydrophobic bases to the matrix
and provides an essentially one step purification of yeast
phenylalanine tRNA starting from total yeast tRNA. This
J. Biosci. 31(4), October 2006
Uttam L RajBhandary and Caroline Köhrer
446
January 21, 1969
Input: 438 OD260
tRNAPhe
Figure 15. BD-cellulose chromatography pattern of crude yeast tRNA. Column: BD-cellulose (1 x 20 cm). Gradient: 0.05 M sodium
acetate pH 5.0, 0.01 M Mg2+ from 0.3 to 1.0 M NaCl. Elution of tRNAPhe with 10% ethanol at 1.0 M NaCl (from: RajBhandary 1969;
personal records).
crude tRNA
partially purified tRNAiMet
pH 7.0, 37°C
pH 4.4, 42°C
Figure 16. Purification of N. crassa mitochondrial initiator tRNA. RPC-5 chromatography pattern of crude mitochondrial tRNA (left),
re-chromatography of partially purified mitochondrial initiator tRNA (right) (from: Heckman et al 1978).
tRNA uniquely contains the hydrophobic base Y that is
exposed and is located next to the anticodon sequence.
Because of this, the yeast phenylalanine tRNA binds tightly
to the column even in 1 M NaCl and can be eluted off the
column only in the presence of 10% ethanol (figure 15). The
easy purification of yeast phenylalanine tRNA and the fact
that it contained a strongly fluorescent base led to its use
in many of the early biophysical and functional studies on
tRNAs by several laboratories including that of Zachau.
Because of the ease of purification, the tRNA also became
commercially available which was an important factor in this
tRNA being the first one whose three-dimensional structure
J. Biosci. 31(4), October 2006
was established, by two laboratories working independently
(Kim et al 1974; Robertus et al 1974). It is also noteworthy
that this tRNA remained the only RNA of known threedimensional structure for several years.
DEAE-Sephadex and Sepharose 4B columns also
proved quite useful for large scale purification of many
tRNAs. RPC columns, which could be run at different
pHs and temperatures and at high pressure, were quite
versatile. Because the columns are run at high pressure,
chromatography is quite rapid allowing for fractionation of
not just tRNAs but aminoacyl-tRNAs. They also provided
excellent resolution of tRNAs on either small scale or
Early days of tRNA research: Discovery, function, purification and sequence analysis
tRNA
Xp….…pGp
….…
T1-RNase
32pXp….…pG
OH
Xp….…pG
….… OH
Phosphatase
447
+ Glucose-6-32P
(i)
Polynucleotide kinase
+ 32pppA
(ii)
Hexokinase
+ glucose
Figure 17. Schematic representation of in vitro 32P-labelling of T1-RNase fragments of tRNA.
large scale. Figure 16 provides an example of a two step
purification of Neurospora crassa mitochondrial initiator
methionine tRNA starting with total mitochondrial tRNA
available only in limited amounts (Heckman et al 1978).
The availability of these purification methods and newer
methods for tRNA sequencing (below) allowed sequence
analysis of tRNAs from eukaryotic organelles such as
mitochondria and chloroplasts.
7.
Use of in vitro 32P-labelling in sequence analysis
of tRNAs
The approach described above for sequence analysis of
P-labelled tRNAs is applicable only to those tRNAs that
can be labelled in vivo with 32P to high specific activity and
that can be purified readily. The next major development
in sequencing of tRNAs involved the use of in vitro 32Plabelling of tRNAs and oligonucleotides derived from
the tRNA. Described below are several approaches that
use in vitro 32P-labelling for sequence analysis of tRNAs
(RajBhandary 1980). Use of these approaches has allowed
sequence analysis of tRNAs available only in small amounts
from eukaryotic organelles, including identification of
modified nucleotides (Chang et al 1976; Heckman et al
1978), and requiring as little as 5–10 µg of tRNA compared
to almost one gram of tRNA in earlier work.
The first approach involves the use of T4-polynucleotide
kinase to label the 5’-ends of oligonucleotides present in
pancreatic or T1-RNase digests of the tRNA with 32P (figure
17). The 5’-end labelled oligonucleotides are separated
using two-dimensional electrophoresis (figure 18) or twodimensional homochromatography (electrophoresis in the
first dimension followed by homochromatography on thinlayer plates of DEAE-cellulose in the second dimension).
Sequence of the oligonucleotides is determined using
the characteristic mobility shifts between the successive
32
Figure 18. Fingerprint pattern of in vitro 32P-labelled tRNA.
T1-RNase products of N. crassa mitochondrial tRNATyr. First
dimension: electrophoresis on cellulose acetate paper at pH 3.5.
Second dimension: electrophoresis on DEAE-cellulose paper in
7% formic acid (from: RajBhandary 1980).
intermediates in a partial 3’-exonuclease digest of the
oligonucleotide (figure 19). Modified nucleotides present at
the 5’-end that carries the 32P-label can be identified directly,
those located internally can only be identified indirectly
(Silberklang et al 1979).
Much of the overlap information necessary for ordering of
the oligonucleotides into a unique sequence can be obtained
by partial digestion of 5’- and 3’-end labelled tRNAs4 with
4
tRNAs were labelled at the 3’-end with 32P using tRNA
nucleotidyl-transferase (the CCA enzyme) and α-32P-ATP.
J. Biosci. 31(4), October 2006
448
Uttam L RajBhandary and Caroline Köhrer
Figure 19. Autoradiography of partial snake venom phosphodiesterase digestion of 5’- 32P-UAUCCG. RNase T1 products of
N. crassa mitochondrial tRNATyr. First dimension: electrophoresis
on cellulose acetate paper at pH 3.5. Second dimension:
homochromatography on a 20 x 20 cm DEAE-cellulose thin-layer
plate (from: RajBhandary 1980).
Figure 21. Autoradiography of partial nuclease P1 digest of 5’labelled N. crassa mitochondrial initiator tRNA. First dimension:
electrophoresis on cellulose acetate paper at pH 3.5. Second
dimension: homochromatography on a 20 x 20 cm DEAE-cellulose
thin-layer plate (from: RajBhandary, 1980).
Figure 20. Use of nuclease P1 in sequence analysis of 5’- or 3’-labelled RNA (from: RajBhandary 1980).
J. Biosci. 31(4), October 2006
Early days of tRNA research: Discovery, function, purification and sequence analysis
nuclease P1, a random endonuclease (figure 20). The 32Plabel containing oligonucleotide products are analysed by
two-dimensional homochromatography and the sequence of
twenty or more nucleotides from the labelled end can often
be determined in a single step (figure 21).
A second approach for sequence analysis involves partial
digestion of 5’- and 3’- 32P-labelled tRNAs with base specific
nucleases (Simoncsits et al 1977). In principle, this approach
is similar to the Maxam and Gilbert method for sequencing
DNA (Maxam and Gilbert 1977) except that base specific
enzymes rather than base specific chemical reagents are used
Figure 22. Schematic representation of RNA sequencing
including direct identification of modified nucleotides in tRNAs
(from: RajBhandary 1980).
449
for partial cleavage of 32P-end labelled tRNAs. The enzymes
used are T1-RNase (G-specific), U2-RNase (A-specific),
an extracellular RNase from Bacillus cereus (C- and Uspecific), a nuclease from chicken liver (C-specific); and
alkali, formamide or T2-RNase for non-specific cleavage.
The 32P-labelled partial digestion products are separated by
denaturing polyacrylamide gel electrophoresis (PAGE) and
the location of G, A, U and C residues relative to the labelled
end provides much of the sequence. Because of the presence
of modified nucleotides including 2’-0-methyl nucleotides,
which are resistant to most nucleases, this approach alone
is not sufficient to derive the complete sequence of a tRNA.
It does, however, provide most of the overlap information
necessary for rapidly assembling oligonucleotides from
complete digests into a unique sequence.
Another approach for tRNA sequencing applicable
to 32P-labelled tRNA involves a chemical method along
the same lines as those used by Maxam and Gilbert for
sequencing DNA. It utilizes four different base specific
chemical reactions followed by cleavage of phosphodiester
bonds at those sites (Peattie 1979). The 3’-end labelled
oligonucleotide fragments are separated by PAGE and
the location of G, A, U and C relative to the labelled end
provides the sequence. This approach complements the base
specific enzymatic digestion method for sequence analysis
of 32P-end labelled tRNA.
A general approach that allows sequence analysis
of a tRNA including direct identification of modified
nucleotides was developed by Stanley and Vassilenko
(1978). It involves hydrolysis of tRNA under conditions
such that hydrolysis is random and partial (figure 22). A
homologous series of oligonucleotides each bearing the
common 3’-end of the tRNA is obtained. The free 5’-OH
group in these series of oligonucleotides are labelled with
Figure 23. E. coli tRNATyr. Autoradiography of T2-RNase digest on a homologous series of 5’-32P-oligonucleotides generated by partial
alkaline hydrolysis followed by 5’-32P-labelling. Thin-layer chromatography on PEI-cellulose in 0.55 M ammonium sulphate (from:
RajBhandary 1980).
J. Biosci. 31(4), October 2006
Uttam L RajBhandary and Caroline Köhrer
450
32
P- and the 5’-32P-labelled oligonucleotides separated by
PAGE. Identification of the 5’-32P-labelled end of each of
the oligonucleotides provides the sequence of the tRNA.
Randerath, Nishimura and their coworkers (Gupta and
Randerath 1979; Kuchino et al 1979) have greatly simplified
the analytical procedure by including the direct transfer of
the 5’-32P-labelled oligonucleotides from the gel onto a
sheet of polyethyleneimine (PEI) cellulose. The 5’-32Plabelled oligonucleotides are digested to completion in situ
with T2-RNase (or nuclease P1) and the 32P-Xp (or 32P-X)
produced identified directly by thin-layer chromatography.
Figure 23 shows a thin-layer chromatographic pattern from
which the sequence of nucleotides –35 to –75 from the 3’end of E. coli tyrosine tRNA can be read directly. Because
this method of tRNA sequencing allows direct identification
of most modified nucleotides, it has become the method of
choice for tRNA sequencing and continues to be used in the
laboratories of Giegé, Kern and coworkers (Dubois et al
2004) and Watanabe, Suzuki and coworkers (Sakurai et al
2005).
Acknowledgements
Work in our laboratories on tRNAs is supported by grants
R37GM17151 and GM67741 from the National Institutes
of Health and grant WP11NF from the US Army Research
Office.
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J. Biosci. 31(4), October 2006