Intramolecular crosslinking of yeast phenylalanine tRNA in aqueous solution with rigid, variable-length crosslinking reagents, which we call "molecular rulers," has given results in reasonable agreement with the crystal structure. Chlorambucilyl-[^(3)H]phenylalanyl-tRNA^(Phe) crosslinked intramolecularly at G-71 and A-73, whereas chlorambucilyl-pentadecaprolyl-[^(3)H]phenylalanyl-tRNA^(Phe) crosslinked at G-20 and Y-37. The pentadecaprolyl reagent was predicted to be 62 Å long, including chlorambucil and phenylalanine; the sites that it reached are 60 Å distant from the 3' OH (in the case of G-20) or 80 Å distant (in the case of Y-37) in the crystal structure of tRNA^(Phe). The close agreement between the length of the reagent and the distance of G-20 from the 3' OH in the crystal structure illustrates the rigidity of the tRNA^(Phe) molecule in the dihydrouridine loop region at the corner of the molecule. The apparent ability of the 62-Å-long reagent to crosslink to a site, Y-37, that is 80 Å distant from the 3' OH in the crystal structure appears to illustrate the flexibility of both the 3' A-C-C-A terminus and the anticodon stem and loop, with respect to the tRNA molecule. These observations demonstrate the utility of oligoproline-based crosslinking reagents as rigid, variable-length molecular rulers for biological macromolecules in solution

Ainpour, Parviz R.

Behlen, Linda S.

Reuben, Michael A.

Wickstrom, Eric

Caltech Authors - Main

Proc. Natl. Acad. Sci. USA
Vol. 78, No. 4, pp. 2082-2085, April 1981
Biochemistry
Molecular rulers for measuring RNA structure: Sites of
crosslinking in chlorambucilyl-phenylalanyl-tRNAPhe -(yeast) and
chlorambucilyl-pentadecaprolyl-mphenylalanylmtRNAPhe (yeast)
intramolecularly crosslinked in aqueous solution*
(tRNA solution structure/alkylation/gel sequence-determinations)
ERIc WICKSTROM, LINDA S. BEHLENt, MICHAEL A. REUBENS, AND PARVIZ R. AINPOUR§
Department of Chemistry, University of Denver, Denver, Colorado 80208
Communicated by Melvin Calvin, December 15, 1980
ABSTRACT Intramolecular crosslinking of yeast phenylala-
nine tRNA in aqueous-solution with rigid, variable-length cross-
linking reagents, which we call "molecular rulers," has given re-
sults in reasonable agreement with the crystal structure.
Chlorambucdilyl-[aHlphenylalanyl-tRNAPhe crosslinked intramo-
lecularly at G-71 and A-73, whereas chlorambucilyl-pentadeca-
prolyl-[ H]phenylalanyl-tRNAPhe.crosslinked at G-20 and Y-37.
The pentadecaprolyl reagent was predicted to be 62 A long, in-
cluding chlorambucil and phenylalanine; the sites that it reached
are 60 A distant from the 3' OH (in the case ofG-20)or 80 Adistant
(in the case of Y-37) in the crystal structure of tRNAPh,. The close
agreement between the' length of.the reagent and the distance of
G-20 from'the 3' OH in the crystal structure illustrates the rigidity
of the tRNAPh, molecule in the dihydrouridine loop region at the
corner of the molecule. The.apparent ability of the 62-A4ong re-
agent to crosslink to a site, Y-37, that is 80 A distant from the 3'
OH in the crystal structure appears to illustrate the flexibility of
both the 3' A-C-C-A terminus. and the anticodon stem and loop,
with respect to the tRNA molecule. These observations demon-
strate the utility of oligoproline-based crosslinking reagents as
rigid, variable-length molecular rulers for biological macromole-
cules in solution.
Remarkable agreement has been observed between the crystal
structure of yeast phenylalanine tRNA. and its biological func-
tion and solution chemistry (1, 2). The generality of that struc-
ture for the function of all tRNA molecules has been noted (3).
On the other hand, substantial questions remain as to the ex-
.istence and significance of other conformations of tRNA in so-
lution, aminoacylated or unacylated, bound to synthetase, or
bound to a ribosome (4-6).
Rigid, variable-length oligoproline crosslinking reagents,
which we call "molecular rulers" (7) are a potentially powerful
tool for probing the solution structures of complex biological
macromolecules or assemblies of macromolecules. The useful-
.ness of known-length oligoprolines as molecular rulers was
demonstrated by Stryer and Haugland (8) in a series. of singlet-
singlet fluorescence energy-transfer experiments. Poly(L-pro-
line) has a trans conformation in solid state and aqueous solution
known as poly(L-proline) II (9). This conformation is particularly
stiff, and the dimensions of poly(L-proline) II in solution ap-
proximate the dimensions.found in the crystalline state (3.12
Aper residue) (9) up to 50 or more residues long (10, 11).
We have chosen to test the feasibility of molecular rulers on
a well-studied model system, yeast phenylalanine tRNA (1, 2),
before applying them to less well-understood structures. The
Oligoproline
Amino o \ CcG02 Chlorambucil
acid V\
34
0 72 \{~7I~~ ~ ~ ~ ~ ~ 2 3
<33~~~~~~~~~~~~~~3
31
FIG. 1. A molecular ruler, chlorambucilyl oligoproline, linked by
a peptide bond to an aminoacyl-tRNA. When this species is dissolved
in aqueous buffer, intramolecular alkylation may occur. The tRNA
structure is-that of yeast tRNAPhe, adapted from Robertus et al. (13).
resolution offered by oligoproline molecular rulers as a tech-
nique for distance measurement in solution also may provide
useful data to help answer the. questions mentioned above con-
cerning the solution structures of tRNA. Our approach, which
is similar to that of Grachev and Rivkin (12) in the case of zero
proline residues, is shown schematically in Fig. 1. High purity
oligoprolines of defined length have been synthesized with 5,
11, and 15 residues (14), modified with chlorambucil at the NH2
terminus, activated at the COOH terminus with N-hydroxy-
succinimide, and then allowed to react with phenylalanyl-
tRNAPhe (7). Modifications of tRNA were carried out in 80%
dimethyl sulfoxide (Me2SO) or 40% Me2SO and 40% CHC13,
conditions under which tRNA deacylation and alkylation were
minimal (7, 15). Phenylalanyl-tRNAPhe modified with chlor-
ambucilyl-oligoproline molecular rulers was then redissolved
in aqueous buffer to activate the chlorambucil and to allow in-
tramolecular crosslinking at sufficient dilution (' 0.1 ,tM) so
* This, is paper 4 in the series "Molecular Rulers for Measuring RNA
Structure." Paper 3 is ref. 7.
t Present address: Department of Chemistry, University of Colorado,
Boulder, CO 80309.
f Present address: Division of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, CA 91125.
§ Present address: Howmedica, Inc., Groton, CT 06340.
2082
The publication costs ofthis article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertise-
ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Proc. Natl. Acad. Sci. USA 78 (1981) 2083
that no intermolecular crosslinking was detected (unpublished
data).
In this paper we report the sites of intramolecular crosslink-
ing observed when molecular rulers with 0 and 15 proline res-
idues were applied to yeast phenylalanine tRNA and discuss the
significance of these results for the solution structure of that
tRNA and the general applicability of the molecular ruler
technique.
MATERIALS AND METHODS
Yeast phenylalanine tRNA (10 .tM; Boehringer Mannheim) was
alkylated bimolecularly with 1.0 mM chlorambucil (Burroughs
Wellcome, Research Triangle Park, NC) in 1.0 mM Mg(OAc)2/
10 mM (EtOH)3N-HCl, pH 8.0, for 1 hr at 370C, as described
(16). Chlorambucilyl-(prolyl)n-[3H]phenylalanyl-tRNAPhe with
n = 0 and 15 were synthesized in 80% Me2SO (for n = 0) or
in 40% Me2SO and 40% CHC13, (for n = 15) to minimize in-
tramolecular crosslinking during the modification reaction (7)
and to maximize the yield (15). Intramolecular crosslinking was
carried out in 1.0 mM Mg(OAc)2/10 mM NH4OAc, pH 6.0, for
4 hr at 37°C, as described (7). Alkylated or crosslinked nucleo-
sides were determined by the rapid gel sequence-determina-
tion technique of Peattie (17), in which the chlorambucil al-
kylation step substituted for the dimethyl sulfate step. Labeling
ofthe 3' OH ofcrosslinked tRNA molecules with [5'-32P]cytidine-
iL -- Oh 4h
PH9 TI N2H4
Ti Chll
S.
G20
m' G26 -2
A24
630
9 Gm 34
Y37
N2 '14
9
0 .....I =.....642G43A44G45mT G46 AmFG46.Al M--G51
U52
G53
T54
G57
657
}
....wPr
U12 G18
G20
D16,17
U33
Y37
Y37
U41
m7G46
U47
C48
m5C49
U50
G51GSI~~~~~~~~~~~~~
i 592 T5.
G53
154
C 56
C57
m'A58
U59
Cra G65
G65
G65
FIG. 2. Autoradiogram of yeast tRNAPhe alkylated bimolecularly
with chlorambucil and then analyzed by rapid gel sequencing (17).
Lanes: T1 and N2 H4, calibration ladders from treating nonalkylated
tRNA with ribonuclease T1 and hydrazine, respectively, after end la-
beling; Chl, yeast tRNAPhe alkylated with chlorambucil before end la-
beling and treatment with NaBH4 and aniline.
9.
FIG. 3. Autoradiogram of chlorambucilyl-[3Hlphenylalanyl-
tRNAPhe, analyzed by rapid gel sequence determination as in Fig. 2.
The sample in lane Oh was not allowed to crosslink in aqueous buffer,
whereas the sample in lane 4h was allowed to crosslink for 4 hr.
b
47
A..
..
...
=...,=
i
74
75
Biochemistry: Wickstrom et al.
.,V
4041-D
..:i-"-'-'i
2084 Biochemistry: Wickstrom et al.
3',5'-bisphosphate (New England Nuclear) (18) was allowed to
proceed for 48 hr to allow deacylation to occur in the labeling
reaction mixture. This eliminated the need for a separate dea-
cylation step. Gels were autoradiographed with Kodak X-Omat
RP film backed by a pair of DuPont Lightning-Plus screens at
-70'C. Densities ofbands on autoradiograms were quantitated
by scanning the films at 580 nm with an ISCO UA-5 detector
equipped with a gel scanning accessory on line to a Columbia
Scientific CSI-38 integrator.
RESULTS
The reaction pattern of bimolecular chlorambucil alkylation of
tRNAPhe is shown in Fig. 2. The more prominent bands appear
to be led by "ghost" bands that are one residue shorter, perhaps
caused by an impurity in the tRNAPhC preparation that lacks the
3' terminal adenosine. Within the limits ofresolution ofthe gel
autoradiogram, the most reactive nucleotides (Fig. 2, lane Chl)
appear to include G-18, G-20, mG-34, and Y-37. Gel autora-
diograms run for shorter times also showed reactive nucleotides
at G-71, A-73, and A-76.
The pattern of intramolecular crosslinking ofchlorambucilyl-
[3H]phenylalanyl-tRNAPhe is seen in Fig. 3. A sample of the
latter tRNA, which had been modified but had not experienced
intramolecular crosslinking in aqueous buffer, was analyzed
(Fig. 3, lane Oh) to observe the effects ofwhatever crosslinking
and bimolecular alkylation had occurred during the modifica-
tion step. [Previously we had observed that about 10% of the
modified tRNA appeared to have been crosslinked during mod-
ification (7)]. The pattern of reactivity (Fig. 3, lane Oh) closely
resembles that for bimolecular chlorambucil alkylation (Fig. 2,
lane Chl). Because liquid chromatography on Sephadex G-100
had indicated a lack of intermolecularly crosslinked tRNA di-
mers (7), and because none were seen on these autoradiograms,
some bimolecular alkylation of tRNAPh' by the N-hydroxysuc-
cinimide ester ofchlorambucil appears to have occurred during
the modification reaction. This is not unreasonable, because we
have found that 80% Me2SO inhibits the alkylation rate by two
orders of magnitude but does not completely abolish it (7). The
fragments from chlorambucilyl-[3H]phenylalanyl-tRNAPhe that
had been allowed to crosslink for 4 hr in aqueous buffer are seen
in Fig. 3, lane 4h. Bands that are significantly darker than those
in lane Oh occur at the positions of G-71 and A-73. These bands
were quantitated by scanning the autoradiograms and compar-
ing the peak areas with that ofm7G46, which acts as an internal
standard in Peattie's technique (17) for detecting alkylated pu-
rines. The ratios of the alkylated nucleotide band areas to the
m7G-46 band area are presented in Table 1.
The reaction pattern of intramolecular crosslinking of chlo-
Table 1. Ratios of intramolecularly crosslinked nucleotide band
areas to the m7G-46 band area
Band area relative to that of m7G-46
0 prolines* 15 prolines*
Nucleotide 0 hrt 4 hrt 0 hrt 4 hrt
G18 0.54 0.45 0.40 0.49
G20 0.53 0.49 0.50 0.87
mG34 0.06 0.08 0.19 0.18
Y37 0.24 0.29 0.83 2.25
G71 0.06 0.38 <0.01 <0.01
A73 0.06 0.21 <0.01 <0.01
A76 0.03 0.04 <0.01 <0.01
* Chlorambucilyl-(prolyl),-[3Hlphenylalanyl-tRNAPhe was synthe-
sized with 0 (n = 0) and 15 (n = 15) prolines.
t Length of time of intramolecular crosslinking in aqueous buffer.
rambucilyl-pentadecaprolyl-[3H]phenylalanyl-tRNAPhe is shown
in Fig. 4, in which a sample of the modified tRNA that had not
been exposed to aqueous buffer after modification (lane Oh) can
be compared with a sample that had been allowed to crosslink
for 4 hr in aqueous buffer (lane 4h). Bands in lane 4h that are
significantly darker than those in lane Oh occur at the positions
of G-20 and Y-37. The quantitation of these bands is shown in
Table 1.
Oh 4h
pH9 T
......
_M
I N2 H4
47
'd;! 1,l:
.
.E3_
:
*3ii;
_::
..
74
75
P 018
G20
S: Y37
m7G46
T55
G65
_T _VF
-1:V1=~~~~~~~
._
-4
FIG. 4. Autoradiogram of chlorambucilyl-pentadecaprolyl-[3H]-
phenylalanyl-tRNAFh', analyzed by rapid gel sequence determination
as in Fig. 2. The sample in lane Oh was not allowed to crosslink in
aqueous buffer, whereas the sample in lane 4h was allowed to crosslink
for 4 hr.
Proc. Natl. Acad - Sci - USA 78 (1981)
W,. =..i
Proc. Natl. Acad. Sci. USA 78 (1981) 2085
DISCUSSION
The sites of reactivity in yeast tRNAPhe for alkylation by chlo-
rambucil agree reasonably well with the results of other chem-
ical modification experiments (19, 20). Our observation of in-
tramolecular crosslinking to G-71 and A-73 by the molecular
ruler with no proline residues is consistent with similar work
by Grachev and Rivkin (12) and with the 15-A length of chlor-
ambucilyl phenylalanine.
Model building and the structure of poly(L-proline) II (9)
predict that chlorambucilyl-pentadecaprolyl-phenylalanine
should be 62 A long. It is of interest to ascertain whether our
observation of crosslinking to G-20 and Y-37 with the latter
molecular ruler is consistent with the observed crystal structure
of yeast tRNAPhe (1, 2). Examination of the atomic coordinates
in the crystal structure (A. Rich, personal communication) in-
dicates that the N-7 of G-20 is 60.1 A distant from the 3' OH,
and that the N-12 of Y-37 is 80.7 A distant.
At first glance, it appears that we have excellent agreement
in the case of G-20 but poor agreement for Y-37. However, for
tRNA in solution, the dihydrouridine loop at the coruer of the
L form is relatively rigid because of tertiary structure interac-
tions (2), whereas the anticodon loop is relatively flexible and
the aminoacyl A-C-C-A terminus is very flexible. The crystal-
structure distance from C-72 to the 3' OH is 19.1 A; flexibility
of this single-stranded region and the relative flexibility of the
anticodon stem and loop with respect to the rest of the tRNA
molecule may account for the ability of the 15-proline ruler to
reach a site in the solution structure of tRNAPhe that is too far
away in the crystal structure. From the point of view of reso-
lution in molecular-ruler distance measurements, it is signifi-
cant that mG-34 (near Y-37 in the anticodon loop) and G-18 (near
G-20 in the dihydrouridine loop) were not significantly
crosslinked.
These results may be viewed from two perspectives. If one
assumes the oligoproline spacers to exist in the poly(L-proline)
II conformation (9), then crosslinking ofthe 62-A-long chloram-
bucilyl-pentadecaprolyl-phenylalanine from the 3' OH to G-20
and Y-37 confirms that the solution structure of yeast tRNAPhe
is reasonably similar to the crystal structure (1, 2). If one as-
sumes in the first place that the crystal structure and solution
structure of yeast tRNAPhe are similar (3), then the crosslinking
results establish the accuracy of oligoproline molecular rulers
for distance measurements.
Note Added in Proof. Since submitting this manuscript, we have found
that chlorambucilyl-pentaprolyl-[3H]phenylalanyl-tRNAPhe crosslinks
intramolecularly to G-45 (unpublished data). Crosslinking of Esche-
richia coli MRE600 ribosomes with chlorambucilyl-[3H]phenylalanyl-
tRNAPhe directed by poly(U) resulted in labeling of50S ribosomal pro-
teins L4, L18-20, and L26-27 (21) and single-strand RNA but not 23S
RNA (unpublished data).
We wish to thank Drs. Debra Peattie and Olke Uhlenbeck for helpful
discussions of gel sequence determinations and end labeling and Drs.
Alexander Rich and Gary Quigley for the exact crystal structure dis-
tances between the 3' OH and our crosslinking sites. This work was
supported by grants from the National Science Foundation (PCM
79-23471) and the National Institutes of Health (GM 24128).
1. Rich, A. (1978) Acc. Chem. Res. 10, 388-396.
2. Kim, S. H. (1979) in Transfer RNA: Structure, Properties, and
Recognition, eds. Schimmel, P. R., Soll, D. & Abelson, J. N.
(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp.
83-100.
3. Clark, B. F. C. (1978) in Transfer RNA, ed. Altman, S. (MIT
Press, Cambridge, MA), pp. 1447.
4. Crothers, D. M. & Cole, P. E. (1978) in Transfer RNA, ed. Alt-
man, S. (MIT Press, Cambridge, MA), pp. 196-247.
5. Ofengand, J. (1977) in Molecular Mechanisms of Protein Biosyn-
thesis, eds. Weissbach, H. & Pestka, S. (Academic, New York),
pp. 8-79.
6. Reid, B. R. (1977) in Nucleic Acid-Protein Recognition, ed. Vo-
gel, H. J. (Academic, New York), pp. 375-390.
7. Reuben, M. A., Ainpour, P. R., Hester, H. L., Neveln, V. L.
& Wickstrom, E. (1981) Biochim. Biophys. Acta, in press.
8. Stryer, L. & Haugland, R. P. (1967) Proc. Natl. Acad. Sci. USA
58, 719-726.
9. Cowan, P. M. & McGavin, S. (1955) Nature (London) 176,
501-503.
10. Steinberg, I. Z., Harrington, W. F., Berger, A., Sela, M. &
Katchalski, E. (1960) J. Am. Chem. Soc. 82, 5263-5279.
11. Schimmel, P. R. & Flory, P. J. (1967) Proc. Natl. Acad. Sci. USA
58, 52-59.
12. Grachev, M. A. & Rivkin, M. I. (1975) Nucleic Acids Res. 2,
1237-1260.
13. Robertus, J. D., Ladner, J. E., Rhodes, D., Brown, R. S., Clark,
B. F. C. & Klug, A. (1974) Nature (London) 250, 546-551.
14. Ainpour, P. R. & Wickstrom, E. (1980) Int. J. Pept. Protein Res.
15, 225-235.
15. Reuben, M. A., Kusnezov, I. J. & Wickstrom, E. (1979) Biochim.
Biophys. Acta 565, 219-223.
16. Wickstrom, E. & Yarus, M. J. (1975) Abstracts of the 170th Na-
tional Meeting of the American Chemical Society, Biological
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R. S., Clark, B. F. C. & Klug, A. (1974) Nucleic Acids Res. 1,
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Biochemistry: Wickstrom et al.


Molecular rulers for measuring RNA structure: sites of crosslinking in chlorambucilyl-phenylalanyl-tRNA^(Phe) (yeast) and chlorambucilyl-pentadecaprolyl-phenylalanyl-tRNA^(Phe) (yeast) intramolecularly crosslinked in aqueous solution

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Molecular rulers for measuring RNA structure: sites of crosslinking in chlorambucilyl-phenylalanyl-tRNA^(Phe) (yeast) and chlorambucilyl-pentadecaprolyl-phenylalanyl-tRNA^(Phe) (yeast) intramolecularly crosslinked in aqueous solution

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