©
1996 Oxford University Press
981-983 Footnote
High yield photocrosslinking of a 5-iodocytidine (IC) substituted RNA to its associated protein
High yield photocrosslinking of a 5-iodocytidine (IC) substituted RNA to its associated protein
Kristen M.
Meisenheimer
,
Poncho L.
Meisenheimer
,
Michael C.
Willis
and
Tad H.
Koch*
Department of Chemistry and Biochemistry, University of Colorado,
Boulder
, CO 80309-0215,
USA
Received November 24, 1995;
Revised and Accepted January 16, 1996
5-Iodouracil is an excellent chromophore for introduction into both RNA and
DNA for the purpose of achieving high yield photocrosslinking of nucleoprotein complexes (
1
,
2
). Crosslinking yields are often three to five times higher than those achieved
with 5-bromouracil, in part, because the 5-iodouracil chromophore absorbs at longer wavelength and consequently
can be excited more selectively. The highest yields are achieved by irradiating
with a helium cadmium laser emitting at 325 nm (
1
); however, useful yields can also be obtained with a 312 nm transilluminator (
1
,
2
). The technique is primarily selective for aromatic amino acid residues;
consequently, success in crosslinking depends upon proximity of a U or T of the
nucleic acid to an aromatic ring of the protein. We now report that 5-iodocytosine is an excellent chromophore for nucleoprotein
photocrosslinking complementing 5-iodouracil.
The nucleoprotein complex selected for demonstrating photocrosslinking with the
5-iodocytosine chromophore was an RNA hairpin within the genome of the
bacteriophage MS2 bound to a dimer of the phage coat protein. The system has
been characterized by a co-crystal structure (
3
), and photocrosslinking has been defined with RNAs bearing 5-iodouridine (
1
) and 5-bromouridine (
4
,
5
) complexed with the almost identical R17 coat protein. Further, the effect of RNA sequence variations on protein binding and
crosslinking is well established (
4
). RNA 1 bearing a single 5-iodouridine at the -5 position (Fig.
1
) binds with high affinity to the coat protein and upon irradiation of the
complex at 325 nm, crosslinks in 75-95% yield to Tyr85 of the coat protein (
1
,
5
).
Figure 1
.
Sequences and secondary structures for three RNA variants of the wild-type RNA sequence which display specific binding to bacteriophage MS2 and
R17 coat proteins. RNAs 2 and 3 were prepared by
in vitro
transcription from synthetic DNA templates by T7 RNA polymerase with [[alpha]-
32
P]ATP and ICTP (Sigma Chemical Co.) substituted for CTP [Milligan, J.F., Groebe,
D.R., Witherell, G.W. and Uhlenbeck, O.C. (1987)
Nucleic Acids Res.,
15
, 8783]. Dissociation constants for complexes of RNAs 2 and 3 with a non-aggregating mutant (5) of the MS2 bacteriophage coat protein are 217 and
107 nM respectively, as determined with a nitrocellulose binding assay (1).
Comparable photocrosslinking is now demonstrated with RNA 2 (Fig.
1
) bearing an iodocytidine at the -5 position and five additional iodocytidines in stem positions.
Irradiation at 325 nm gives 75-95% crosslinking after 2 h with only traces of RNA cleavage. A time
course of the crosslinking is shown by the electrophoretic gel in Figure
2
. The high yield is dependent upon a saturating protein concentration. With a
substantial excess of coat protein but at a concentration sufficient to bind
only 36% of the RNA throughout the irradiation, a maximum crosslinking yield of
only 20% is achieved even after 3 h of irradiation. The balance of the RNA
appears in the electrophoretic gel (not shown) primarily as unreacted RNA (40%)
and two RNA cleavage products (40%). Further, gel electrophoretic analysis of
the reaction mixture from irradiation of the RNA in the absence of coat protein showed no high molecular weight bands and in particular no bands at
the location of the crosslinked nucleoprotein (not shown). This control experiment eliminates the possibility that the
crosslinking band shown in Figure
2
represents an RNA dimer.
Figure 2
.
Polyacrylamide gel electrophoretic analysis of the photochemical experiments.
RNAs 2 and 3 prepared as described in the legend to Figure 1, were each heated
in water to 95o C for 3 min and quickly cooled on ice before use to ensure that the RNAs
were in a hairpin conformation [Groebe D.R. and Uhlenbeck, O.C. (1988)
Nucleic Acids Res.,
16
, 11725]. Prior to irradiation, a 500 [mu]l solution which contained 10 nM RNA 2 or RNA 3, 1 [mu]M MS2 coat protein non-aggregating mutant (5), 5 mM dithiothreitol, 100 mM Tris-HCl (pH 7.5 at 4o C), 80 mM KCl, 10 mM magnesium acetate and BSA (100 [mu]g/ml) was incubated on ice for ~30 min. The solution of the resulting
nucleoprotein complex was then irradiated in a 4 mm wide by 1 cm methacrylate
cuvette at 4o C with an Omnichrome helium cadmium laser emitting 35 mW at 325 nm. The
laser beam diameter was 3 mm. Samples were removed after 0, 1, 2 and 3 h of
irradiation, heated to 95o C for 5 min in 80% formamide/10 mM EDTA (pH 8.0)/1 mg/ml bromophenol blue/1
mg/ml xylene cyanol, and electrophoresed at 400 V through a 20% urea,
polyacrylamide, denaturing gel in 89 mM Tris-borate/2 mM EDTA (pH 8.0).
The nature of the photocrosslink at a molecular level is suggested by the
structure of the adduct from 325 nm irradiation of 5-iodocytidine with
N
-acetyltyrosine
N
-ethylamide. The adduct was isolated and characterized from NMR and mass
spectral data as
N
-acetyl-m-(5-cytidinyl)tyrosine
N
-ethylamide with the crosslink connecting the 5-position of cytidine to a meta position of the tyrosine derivative
as shown in Figure
3
.
Figure 3
.
The structure for the adduct from irradiation of 5-iodocytidine (Sigma Chemical Co.) with
N
-acetyltyrosine
N
-ethylamide [Dietz, T.M. and Koch, T.H. (1987)
Photochem. Photobiol.
46
, 971] at 325 nm as established from the following NMR and mass spectral data:
1
H NMR (400 MHz, DMSO-
d6
) [delta] 0.95 (t, J = 7, NHCH
2
C
H
3
), [delta] 3.03 (dq, J = 5,7, NHC
H
2
CH
3
), [delta] 7.94 (t, J = 5, N
H
H
H
H
H
H
H
)
m/z
490, FAB+
m/z
492, (calculated M+H 492). The assignment of the ribose proton signals was
established from an HH COSY spectrum; ribose hydrogens appear as quartets due
to similar coupling with three non-equivalent neighbors.
Although specificity in photocrosslinking for the -5 position in this system was defined earlier with RNAs bearing 5-bromouridine at multiple sites (
4
), it is confirmed here by a second experiment with RNA 3 bearing a uridine at
the -5 position and ICs only in stem positions (Fig.
1
). Irradiation of RNA 3 at a saturating protein concentration (Fig.
2
) or with the RNA 50% bound to coat protein (data not shown) for 3 h with 325 nm
irradiation gives no photocrosslinking and little or no apparent photodamage to
the RNA.
The experiments with RNAs 2 and 3 demonstrate simultaneous photoreactivity and
photostability of the same chromophore at multiple sites within a nucleoprotein complex. The result suggests that photoreactivity is a function of the local environment. The co-crystal structure of an analogous complex (
3
) locates the pyrimidine at the -5 position in a [pi]-stacking arrangement with Tyr85 and none of the bases at stem
positions in proximity with aromatic amino acid residues. Interestingly, a
pyrimidine-tyrosine [pi]-stacking arrangement also appears at the crosslinking site of
U1 snRNA with the N-terminal RNA binding domain of human U1A snRNP protein (
2
,
6
). In the absence of coat protein, another base of RNA 2 may interact with the
IC at the -5 position to promote photocleavage. NMR data for a related RNA in the
absence of coat protein shows the pyrimidine at the -5 position in proximity to the A at the -7 position (
7
).
The possibility of substituting wild-type bases of nucleic acids with either 5-iodouracil or 5-iodocytosine increases the probability of achieving selective,
high yield nucleoprotein photocrosslinking. Although the work reported here has
focused on irradiations with a HeCd laser because of the advantage of
monochromatic 325 nm light, our experience with the IU chromophore (
1
) suggests that useful yields of crosslinking with the IC chromophore can also
be obtained with the broader emission from a 312 nm Transilluminator or
Stratalinker. Crosslinking with either IC or IU appears to be selective for aromatic amino acid residues; from a host guest perspective,
location of a nucleic acid base in a [pi]-stacking arrangement with an aromatic amino acid residue at the
nucleic acid-protein interface seems probable. As a consequence, iodopyrimidine
photocrosslinking of nucleoprotein complexes may be a generally useful
technique for establishing some specific contacts and for locating unknown
proteins suspected of binding to wild-type nucleic sequences.
ACKNOWLEDGEMENTS
This work was supported by the Council for Tobacco Research, USA, The University
of Colorado RNA Center, Nexstar Pharmaceutic Inc., and the National Science
Foundation. T.H.K. also thanks the University of Colorado Council on Research
and Creative Work for a Faculty Fellowship. The authors thank Hans Johansson,
Linda Behlen and Olke Uhlenbeck for DNA templates and samples of non-aggregating MS2 bacteriophage coat protein.
REFERENCES
1 Willis , M.C. , Hicke, B.J., Uhlenbeck, O.C., Cech, T.R. and Koch, T.H. (1993 ) Science 262 1255 .MEDLINE Abstract
2 Stump , W.T. and Hall, K.B. (1995 ) RNA
3 Valegard , K , Murray, J.B., Stockley, P.G., Stonehouse, N.J. and Liljas, L. (1994 ) Nature 371 , 623.
4 Gott , J.M. , Willis, M.C., Koch, T.H. and Uhlenbeck, O.C. (1991 ) Biochemistry 30 6290 .MEDLINE Abstract
5 Willis , M.C. , LeCuyer, K.A., Meisenheimer, K.M., Uhlenbeck, O.C. and Koch, T.H. (1994 ) Nucleic Acids Res 22 4947 . MEDLINE Abstract
6 Oubridge , C. , Ito, N., Evans, P.R., Teo, C.H. and Nagai, K. (1994 ) Nature 372 432 . MEDLINE Abstract
7 Borer , P.N. , Lin., Y., Wang, S., Roggenbuck, M.W., Gott, J.M., Uhlenbeck, O.C. and Pelczer, I. (1995 ) Biochemistry 34 6488 .MEDLINE Abstract
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*
To whom correspondence should be addressed
