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Proc. Natl. Acad. Sci. USAVol. 74, No. 11, pp. 4900-4904,
November 1977Biochemistry
Sequence of an oligonucleotide derived from the 3' end of eachof
the four brome mosaic viral RNAs
(aminoacylation/tRNA-like structure)
RANJIT DASGUPTA AND PAUL KAESBERGBiophysics Laboratory of the
Graduate School and Biochemistry Department of the College of
Agricultural and Life Sciences, University of Wisconsin,
Madison,Wisconsin 53706
Communicated by Heinz Fraenkel-Conrat, August 29, 1977
ABSTRACT A -3'-terminal oligonucleotide fragment, 161bases long,
can be obtained from each of the four brome mosaicvirus BNAs by
means of nuclease digestion. Like the four intactbrome mosaic virus
RNAs, each fragment accepts tyrosine in-a reaction catalyzed by
wheat germ aminoacyl-tRNA synthetase.The complete nucleotide
sequence of the RNA 4 fragment hasbeen determined by use of
standard radiochemical methods.Comparative data for the fragments
from RNAs 1, 2, and 3 showthat-the have nearly the same sequence as
the RNA 4 fragment.The eight bases adjacent to the 3' terminus of
the RNA 4 frag-ment are identical in sequence to the eight terminal
bases oftyrosine tRNA from Torula utilis and eleven interior bases
areidentical in sequence to eleven bases encompassing the
anti-codon region of tyrosine tRNA from Saccharomyces cerevisiae,T.
utilis, and Escherichia coli. Nevertheless, reasonable base-pairing
schemes yield, at best, a distorted cloverleaf
secondarystructure.
Nucleotide sequence analysis of the extremities of plant
viralnucleic acids has provided substantial information
regardingtheir structure and their function as messengers. For
example,the 5'- termini of some of these RNAs, in common with
mosteukaiyotic messengers, contain the "cap" structure
m7GpppGp.(For an extensive review, see ref. 1.) The efficiency of
transla-tion is dependent on the presen'ce of cap (2-4). For
bromemosaic virus (BMV) RNA 4, the monocistronic messenger forBMV
coat protein, the initiation codon is only 10 nucleotidesfrom the
capped 5' end and this short sequence, together withthe cap,
constitutes an effective ribosome binding site (5).iThe 3' termini
of some plant viral RNAs have the unique
property among messengers that they can quantitatively acceptan
amino acid in a reaction catalyzed by aminoacyl'tRNAsynthetase.
Thus, turnip yellow mosaic virus RNA (6) and eggplant mosaic virus
RNA (7) can be charged with valine; tobaccomosaic virus RNA can be
charged with histidine (8). Each ofthe four BMV RNAs can be charged
with tyrosine (9). Somepicornaviral RNAs can be charged, albeit
inefficiently andprobably only after they have been fragmented (10,
11). Theproperty of chargeability implies that these RNAs have
atRNA-like structure and possibly also a tRNA-like function (12).A
secondary structure, somewhat like that of tRNA, is com-patible
with the sequences determined for the 3'-end regionsof turnip
yellow mosaic virus RNA and possibly egg plantmosaic virus RNA (13,
14). The nucleotide sequence at the 3'-terminal region of tobacco
mosaic virus RNA seems less ame-nable to folding into a cloverleaf
secondary structure (15).
Although the BMV RNAs can be aminoacylated with tyro-sine, this
amino acid is not donated to nascent peptides uponin vitro
translation (16).> However, integrity of the 3' end is
necessary for infectivity of BMV RNA (17). Thus, like their
5'ends, the 3' ends of these viral RNAs have a distinctive
structureand presumably an important, although unknown, function.We
have reported that partial hydrolysis of each of the four
BMV RNAs with RNase T1 releases a 3'-terminal fragmentabout 160
nucleotides long-and that this fragment is virtuallyas efficient in
accepting tyrosine as the intact BMV RNAs (18).The present paper
reports the complete nucleotide sequenceof the fragment derived
from RNA 4 and gives data indicatingthat the fragments from the
other three RNAs have nearly thesame sequence. They are each 161
bases long, and we designatethem Q161 of BMV RNAs 1, 2, 3, and
4.
MATERIALS AND METHODSMaterials. T1, T2, and U2 RNases were
obtained from
Calbiochem. Pancreatic ribonuclease, snake venom
phospho-diesterase, and bacterial alkaline phosphatase were
obtainedfrom Worthington Biochemical Corp. Nuclease P1 was
ob-tained from Yamasa Shoyu Co., Ltd. (Tokyo, Japan). T4
poly-nucleotide kinase was obtained from P-L Biochemicals,
['y-32P]ATP, at a specific activity of 1500-2000 Ci/mmol,
wasobtained from Amersham/Searle Corp. Nuclease S1, purifiedby the
method of Vogt (19), was a gift from James E. Dahlbergof the
University of Wisconsin. Thin-layer plates (CEL 300DEAE or CEL 300
DEAE/HR-2/15) were purchased fromMacherey-Nagel and Co.
Preparation of BMV RNA and Fragment Q161. The pro-cedures for
growth and radioactive labeling of BMV and iso-lation and
fractionation of its RNAs have been described(20-22). Partial
digestion of the individual BMV RNAs withRNase T1 and subsequent
isolation of Q161 by electrophoresison polyacrylamide gels have
been described (18). Except whereexplicitly noted, all our
descriptions refer to Q161 isolatedseparately from each of the four
RNAs.
Sequence Analyses. Complete digestion of 0161 with eitherT1
RNase or pancreatic ribonuclease, separation of the
resultingoligonucleotides by two-dimensional electrophoresis or
byelectrophoresis-homochromatography, and further analysis ofthese
oligonucleotides were according to the methods of Sangerand his
colleagues (23, 24).The larger oligonucleotides, especially those
rich iii pyri-
midines, were also analyzed by the wandering spot procedureas
described by Silberklang et al. (13). Oligonucleotides werelabeled
at their 5' ends with 32P by use of [y-32P]ATP andpolynucleotide
kinase according to the procedures of Simseket al. (25).T1 and
pancreatic ribonuclease oligonucleotide catalogs were
obtained for a variety of large and small pieces of Q161,
espe-cially those having overlapping sequences. To obtain large
Abbreviation: BMV, brome mosaic virus.
4900
The costs of publication of this article were defrayed in part
by thepayment of page charges. This article must therefore be
hereby marked"advertisement" in accordance with 18 U. S. C. §1734
solely to indicatethis fact.
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Proc. Natl. Acad. Sci. USA 74 (1977) 4901
,S P21 P19 +P20O
VP
P15P16 *P14 1PI2
P12SPIN PIO
* * P8*7
* P6
P5 P4T2
*PIPi!
FIG. 1. Autoradiograms of 1161 digests. All oligonucleotides
wereanalyzed by pancreatic, T1, U2, and snake venom (preceded by
al-kaline phosphatase) ribonucleases and some also by the
wanderingspot technique. (Left) Two-dimensional fractionation of a
completeT1 RNase digest of 1161. The first dimension was
electrophoresis oncellulose acetate at pH 3.5; the second dimension
was homochroma-tography on a DEAE-cellulose (DEAE/HR 2/15)
thin-layer plate. Thedotted circle marked B indicates the position
of the blue dye. Notethat the oligonucleotide A-C-C-AOH (spot T8)
is readily identifiablefrom its unusual position on the
fingerprint. The spots T1-T26 andtheir relative molar yield are:
T1, Gp, 8.4; T2, C-Gp, 1.2; T3, A-Gp,3.8; T4, C-A-Gp, 0.8; T5,
A-A-Gp, 0.9; T6, C-A-A-Gp, 1.1; T7, A-C-A-C-Gp, 0.7; T8, A-C-C-AOH,
0.8; T9, U-Gp, 4.0; T10, C-A-U-Gp, 1.0;Til, U-A-C-C-Gp, 0.8; T12,
U-A-C-A-Gp, plus T13, C-A-U-A-Gp,2.0; T14, A-A-U-Gp, 1.0; T15,
U-U-Gp, 1.1; T16, C-U-U-Gp, plus T17,U-U-C-Gp, 2.0; T18,
U-C-U-A-Gp, 1.2; T19, A-A-A-A-A-C-A-C-U-Gp, 0.9; T20,
A-A-C-C-C-U-U-A-Gp, 0.9; T21, A-C-C-U-C-U-U-A-C-A-AGp, 1.0; T22,
U-A-A-A-U-C-U-C-U-A-A-A-A-Gp, 1.1; T23,C-U-C-U-C-U-U-C-Gp, 0.9;
T24, C-C-U-U-U-Gp, 1.1; T25, U-C-U-U-A-Gp, 0.8; and T26,
U-U-A-C-U-C-U-U-U-Gp, 1.0.
(Right) Two-dimensional fractionation of a complete
pancreaticribonuclease digest ofQ161. The oligonucleotides were
separated byelectrophoresis in the first dimension on cellulose
acetate at pH 3.5and in the second dimension on DEAE-cellulose
paper in 7% formicacid. The spots Pl-P21 and their molar yield are:
P1, Up, 28.7; P2,Cp, 16.6; P3, A-Cp, 7.5; P4, G-Cp, 5.2; P5, A-Up,
1.9; P6, A-G-Cp, 0.9;P7, A-G-A-Cp, 1.0; P8, A-A-A-Up, 0.9; P9,
G-Up, 5.0; P10, G-A-A-A-A-A-Cp, 0.7: P11, A-G-Up, 1.2; P12,
A-A-G-Up, 1.1; P13, A-G-A-A-Up, 1.0; P14, G-G-G-Cp, 0.9; P15,
A-A-A-A-G-A-G-A-Cp, 0.9; P16,A-G-G-Up, 1.0; P17, A-A-G-A-G:-Up,
1.2; P18, G-G-A-A-G-A-A-Cp,0.9; P19, G-A-G-A-G-Up, 0.9; P20,
G-G-G-Up, 0.9; P21, A-G-G-G-G-Up, 0.5.
overlapping pieces, 0161 was digested with 20 ng of
pancreaticribonuclease or 400 ng of T1 RNase per 200 jig of RNA.
Theincubation was at 00 for 20 min. To obtain comparativelyshorter
pieces (10-30 nucleotides), Q161 was digested with T1or pancreatic
ribonuclease at an enzyme to substrate ratio of1:100. The
incubation was at 40 for 10 min. The 0161 fragmentwas tested for
the presence. of abnormal bases by two-dimen-sional thin-layer
chromatography on cellulose as described byNishimura (26).
Conditions for digesting 0161 with nucleaseS1 were similar to those
of Rushizky and Mozejko (27).
RESULTSLimited digestion of BMV RNA 4 with T1 RNase and
subse-quent fractionation by electrophoresis on polyacrylamide
gelsproduces one major band and many minor bands correspondingto
fragments of various lengths (18). The major band
fragment,designated 0161, is the only one produced quantitatively,
andit can be obtained readily in pure form. On complete
digestion
with RNase T1, this fragment gives rise to the 3'-terminal
oli-gonucleotide A-C-C-AoH, indicating that 0161 is cleaved fromthe
3' end of RNA 4.Sequence Analysis of RNase T1 and -Pancreatic
Ribonu-
clease Oligonucleotides of RNA 4 (161. Fragment 0161 ofBMV RNA
4, uniformly labeled with 32p (specific activity 109dpm/mg of RNA),
was digested to completion with RNase T1and the products were
separated and characterized. A two-dimensional separation by
cellulose acetate electrophoresis andDEAE-cellulose thin-layer
homochromatography is shown inFig. 1 left. All products were well
resolved except the isomersT12 and T13 and T16 and T17. Separation
of similar qualityis obtainable by two-dimensional electrophoresis
(18). Fig. 1right illustrates complete separation by
two-dimensionalelectrophoresis of the products of digestion of 0161
by pan-creatic ribonuclease. The sequences of most of the
pancreaticribonuclease and of the short T1 RNase oligomerswere
deter-mined by analysis with the complementary RNase T2 RNase,snake
venom phosphodiesterase, and U2 RNase. The oligomersfor which such
analyses were not definitive were examined, inaddition, by the
wandering spot procedure; these includedT17-T26 and P15, P17, P18,
and P21. Some typical wanderingspot analyses, those of oligomers
T21, T24, T26, and P18, areshown in Fig. 2. Fragment 0161 of BMV
RNA 4 was digestedwith a mixture of ribonucleases and the products
were analyzedfor the presence of unusual bases. No spotswere
detected otherthan those of the common nucleotides A, C, G, and
U.
Ordering of Oligonucleotides and the Sequence of RNA4 0161.
Fragment Q161 of BMV RNA 4 was partially digestedwith T1 or
pancreatic ribonuclease and the pieces were sepa-rated by
polyacrylamide gel electrophoresis or by celluloseacetate
electrophoresis and homochromatography. The purifiedproducts were
analyzed as had been 0161 itself. Enough piecesof different chain
lengths and with overlapping sequences wereobtained to permit
ordering of all Ti and pancreatic oligonu-cleotides except those
between residues 70 and 74. We couldnot distinguish between the
sequences C-A-U-G-G-G-C-U-U-G-C-A-U-A-Gp and
C-A-U-G-C-U-U-G-G-G-C-A-U-A-Gp, since both sequences gave rise to
the same products aftera variety of partial digestions with T1 and
pancreatic ribonu-clease. This ambiguity was resolved by 32P
end-labeling of theTi partial product corresponding to residues
69-74, followedby wandering spot analysis as shown in Fig. 3.
161 150 140A -C-A-C-G-C-A-G-A-C-C- U -C-U-U-A-C-A-A-G-A- G-
U-G-U-C-U-A-G-G-U-130 120 110 100G -C-C-U-U-U-G-A-G-A- G
-U-U-A-C-U-C-U-U-U- G -C-U-C-U-C-U-U-C-G- G
90 80 70A-A-G-A-A-C-C-C-U- U -A-G-G-G-G-U-U-C-G- U
-G-C-A-U-G-G-G-C-U- U -G-
60 50 40C-A-U-A-G-C-A-A- G -U-C-U-U-A-G-A-A-U- G
-C-G-U-A-C-C-G-G-G- U -G-U-
30 20 10A-C-A-G-U-U-G- A -A-A-A-A-C-A-C-U-G- U
-A-A-A-U-C-U-C-U-A- A -A-A-G-A-G-A-C-C-A-OH
Sequences of 0161 from BMV RNAs 1, 2, and 3. The Tiand
pancreatic ribonuclease maps for Q161 from RNAs 1, 2,and 3 were
identical to those of RNA 4 except as follows: (a) In0161 from RNAs
1 and 2, spot 11 (U-A-C-C-Gp) was missingand was replaced by spots
corresponding to the oligomers U-Gpand U-C-Gp in RNA 1 and U-Gp and
C-C-Gp in RNA 2. (ii)A-Cp (spot P3) was reduced in amount while
G-Up (spot P9)was increased for RNA 1 and G-Cp (spot P4) was
increased forRNA 2. Other possible differences are not precluded,
e.g., se-quence isomers that migrate identically both in
electrophoresisand homochromatography. Because extensive
differences ofthis kind are unlikely and would, furthermore, be
unlikely tohave a bearing on our conclusions, we chose, for the
present,to ignore these possibilities.
T212@9 OT19* T21 @T20T26 S
T23
T25%T24 T13 *T7
T14#p *T6T16+TI70 Te T5T5T
,#WT15 OT4
T9 *T3 TO
Biochemistry: Dasguipta and Kaesberg
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4902 Biochemistry: Dasgupta and Kaesberg
c
\c
U
._
\G0
-.B_m* G\GA).*A I
IA.r
IAjc
FIG. 2. Autoradiogram of partial snake venom
phosphodiesterasedigests (Upper left and right) and partial
nuclease P1 digests (Lowerleft and right) of 5'-32P-labeled spots
T21 (Upper left), T24 (Upperright), T26 (Lower left), and P18
(Lower right) of Fig. 1. CompleteT1 and pancreatic ribonuclease
digests of 0161 were labeled with[,y-32PJATP using polynucleotide
kinase and the products were sep-arated. 5'-32P-Labeled spots were
then eluted, partially digested withthe above enzymes, and
analyzed. The first dimension was electro-phoresis on cellulose
acetate, pH 3.5; the second dimension washomochromatography on
thin-layer plates made of either pureDEAE-cellulose (CEL 300 DEAE;
Upper left and right) or a mixtureof DEAE-cellulose and cellulose
in the ratio 2:15 (CEL 300 DEAE/HR2/15; Lower left and right). The
homochromatography mixture usedwas a 3% solution of yeastRNA in 7M
urea that had been hydrolyzedfor 45 min, prepared according to
Barrell (24). The mononueleotideof T26 waS partially trapped in the
paper wick.
In all probability RNA 4 (161 and RNA 3(161 are identical,RNA 2
(161 differs only in that base 46 is G, and RNA 1 1161differs in
that base 46 is G and base 45 is U.
Sequence Similarity to tRNAs. The susceptibility of q161to
aminoacylation with tyrosine suggests a structural similarityto
tyrosine tRNA. Is this resemblance reflected in the (161sequence?
Comparison shows that only a limited correspon-dence to authentic
tyrosine tRNAs exists. The sequence of theseven and eight bases
adjacent to the 3' terminus of (161 isidentical to that of the
corresponding bases of Saccharomycescerevstsae and Torula utilis
tyrosine tRNA, respectively (28).Also eleven bases in the sequence
of (161, residues 14-24, havethe same sequences as eleven bases in
the anticodon loop regionof S. cerevisiae, T. utilis, and
Escherichia coli tyrosine tRNAexcept that in these tRNAs, three of
the bases are modified.However, the eleven corresponding bases are
not in sequentially
A- Uc
\G
\G
.!,
-~~~~~~~~~~~~~~~~~~~~~~~~~~~-k1:;".~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Il
2.. 3
oI*I*C/A
4A
FIG. 3. Autoradiogram of a partial nuclease P1 digest on a
5'-32P-labeled RNA fragment obtained by partial T1 RNase
digestionof 0161. Partial T1 digestion was done on a mixture of
uniformly32P-labeled 0161 (to aid in locating the bands) and
unlabeled 0161and the products were separated on 10% polyacrylamide
gels. Cor-responding bands were then eluted, labeled with kinase at
their 5' end,and purified further by two-dimensional
electrophoresis and homo-chromatography.
similar locations with respect to their 3' ends. Thus,24 14
1
n 161 A-C-U-G-U-A-A - A-U-C-U...A-G-A-G-A-C-C-AT. utilis Tyr
tRNA . . A-C-U-G-* -A-i6A-A-' -C-U ... .A-G-A-G-A-C-C-A
46 36 1
Other sequence correspondences to Tyr tRNAs are five baseslong
or less.
Secondary Structure and Base Pairing. Secondary structureis not
obviously revealed by inspection of base sequence. Nev-ertheless
some inferences can be drawn by considering struc-tures that
maximize the number of Watson-Crick base pairs.For RNA 4 Q161, the
secondary structure of highest stabilityaccording to the rules of
Tinoco et al. (29) is-shown in Fig. 4.As indicated in that figure,
all but one T1 RNase cleavage pointsof high susceptibility are
located in the regions lacking basepairs. The single exception is
between the C-A bond in positions77-78. A number of essentially
equivalent base-pairing schemesexist. For example, the sequence
A-A-G-A-G-A in positions 4-9can pair with U-C-U-C-U-U in positions
103-108. X-ray crys-tallography shows that in tRNAs the classical
cloverleaf modelaccurately reflects actual base-pairing (although
additional baseinteractions exist that are not revealed by
cloverleaf folding).For 1161, can a cloverleaf structure be drawn
with an accessibleA-C-C-A terminus and a tyrosine anticodon
centered on an"anticodon" loop? Both features are believed to be
involved inrecognition by the charging enzyme (30). With some
alternativefolding and with elimination of some marginal base pairs
thesecondary structure of Fig. 4 can be converted to the
morecloverleaf-like structure shown in Fig. 5. However, we
areunable to construct a secondary structure of high stability
thatprovides an "anticodon" loop centered on 1161 bases 14-24.
It is possible, of course, that tertiary interactions,
unrevealedby cloverleaf base pairs, determine the charging enzyme
rec-ognition features. Regardless of detailed knowledge of
tertiarystructure, it is to be expected that an enzyme recognition
sitewould be near the surface of a substrate molecule and thus
GA'C
/Cx
_
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Proc. Natl. Acad. Sci. USA 74 (1977) 4903
AC145 UCU A C UUA A UCG *U UUA - UGCGu GCCG AUAU GC
N GC z AU"k AUX GCC AGGUGCCUUU U
,.,tG UC CA \ G
A U AG161A 90U CCCAAGAIPA J-1d-Isot
A1c
AGAGA A1AUCUCUA
A it
AUGUCACAA28UACAGUG
G>X AA
G
,C
A47, A GGGUUC G
G UGCGCG
-AU /UAAG
G A55GCUU NkGU \CGUAUAGC
C GA UA66
FIG. 4. Possible secondary structures for RNA 4 Q161 drawn
tomaximum base-pairing. Preferred sites of action of T1 and
pancreaticRNases are indicated by solid and broken arrows,
respectively.
might be especially vulnerable to nuclease attack. The U-Abond
in the "anticodon" region of the structure in Fig. 5 (po-sitions
66-65) is the most susceptible pancreatic ribonucleasepoint in
Q161. With an enzyme to substrate ratio of 1:10,000,it was possible
to obtain a break only at this point in Ql161,separating the
molecule into two parts. The isolated fragmentswere no longer
chargeable. However, a molecule with a hiddenbreak at this point,
with the halves still noncovalently bound,was chargeable (M. Bastin
and P. Kaesberg, unpublished ob-servations), indicating that the
structure on both sides of this
145AC
U A
UA 118CG UUA U ACGU GCCG AU AAU GC CGC AU CAU G UUGCU A
C AGGUGCCUUU CGG UAC CGA UAC U^AAUUA CUAAAU 15
161 G UCG A CAAA AUGUCA A
U A UACAGU A90U CCCAAG GU63A GGGUUC U G
G G GGU U C-
G GGC UA AUC U GU G GA CG UC 50
UA GUA
G CC 6AU 65
FIG. 5. Slightly modified secondary structure for RNA 4
Q161drawn to illustrate a similarity to the cloverleaf structure of
tRNA.
0
0
>: ~~~~~~~~~~dF> I
b
~~i ~ a C
0 20 40 60 80 100FRACTION NUMBER
FIG. 6. Polyacrylamide gel pattern of RNA 4 Q161 that has
beendigested with nuclease S1. The digest was precipitated with
ethanol,dissolved in buffer, and then applied to a 10%
polyacrylamide gel. Theelectrophoresis was carried out at 3 mA for
2.5 hr, at which time thebromophenol dye had moved 10 cm. The gel
was then crushed in aGilson automatic gel crusher. Fraction 1
corresponds to the bottomof the gel.
anticodon-like feature, although not the integrity of the
anti-c6don itself, is necessary. This is similar to the situation
in sometRNAs in which a hidden break in the anticodon loop does
notpreclude aminoacylation (30).
Digestion of Q161 by Si Nuclease. S1 nuclease preferen-tially
cleaves in the anticodon loop of tRNAs (31). It is possibleto
digest tRNA to yield more than 90% half-molecules, that arestable
to 50-fold higher concentration of enzyme (27). We thusundertook a
study of the digestion of Q161 to ascertain whethera similar
preferential cleavage site existed. (161 was digestedwith nuclease
S1 under conditions optimal for cleaving tRNAsin their anticodon
loops. After digestion, the products werefractionated on 10%
polyacrylamide gels. Fig. 6 shows an ex-periment in which 30,000
cpm of 32P-labeled 2161, togetherwith 50Mg of unlabeled yeast RNA,
were digested with 80 unitsof enzyme in 0.3 M NaCl, 0.05 M sodium
acetate (pH 5.7) for24 hr at 00 in a 50-,gl volume. Three major
bands, designatedI, II, and III, and several minor bands were
observed. The RNAfrom each band was eluted and subjected to
electrophoresis andhomochromatography after complete digestion with
RNase T1.The bands were identified by comparison of their T1
catalogswith those of 161. Band I contains all Ti oligonucleotides;
itis undigested 161. Band II contains, in equimolar yield, all
Tioligonucleotides on the 5' side of oligonucleotide T13
(C-A-U-A-Gp) and is thus a cleavage product encompassing
residues69-161. Band III contains (i) in equimolar amount all
Tioligonucleotides on the 3' side of oligonucleotide T13 exceptT8,
(ii) T8 in less than equimolar amount, and (iii) small oli-gomers,
in less than equimolar amount, which we believe areportions of T13
or T8. Band III is thus heterogeneous; it containsresidues 1-68,
but residues 1-4 and 63-68 exist only fractionally.We believe that
minor bands a, b, c, and d represent mixturesof SI cleavage
products. Bands a and b contained most T1oligonucleotides except
C-A-U-A-Gp, A-C-C-AOH, and A-C-A-C-Gp. Bands c and d also contained
most T1 oligonucleotides,but those near the 5' and 3' ends of Q1i61
were present in lowamounts. We conclude that S1 nuclease cleaved
preferentiallynear residue 66, the "anticodon" region of Fig.
5.
DISCUSSIONEarlier publications and this study have shown that
the fourBMV RNAs can be enzymatically aminoacylated with
tyrosine
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4904 Biochemistry: Dasgupta and Kaesberg
in a manner similar to that of tyrosine tRNA, that a
highlysusceptible ribonuclease T1 cleavage site exists 161 bases
fromtheir 3' terminus, and that in each case the 3'-cleavage
product(0161) can be aminoacylated. Moreover, 0161 is
relativelyresistant to nuclease digestion; it is obtained in almost
quanti-tative yield over a wide range of TI concentrations. The
0161molecules from each of the four RNAs have nearly the
samenucleotide sequence. These facts suggest that the 3' end of
theBMV RNAs has a structural resemblance to tyrosine tRNA, thatit
is a tightly folded structure substantially retaining its
con-figuration after cleavage, and (from its sequence
conservation)that it plays an important role in the life cycle of
the virus.
It was thus gratifying, at least for a short time, that the
twolongest sequence identities among S. cerevisiae, T. utilis,
andE. coli tyrosine tRNA, namely, their acylating terminus
and(ignoring base modifications) their anticodon loop region,
ex-isted also in 0161. However, these two regions of sequence
wereseparated from each other by 28 bases in the tRNAs but by
onlyfour in 0161 and, equally perversely, no manner of
stablebase-pairing could produce a structure resembling a
tRNAanticodon loop and stem. However, folding of Q161 into
acloverleaf-like structure provides a stem and loop structure
inwhich anticodon AUA (bases 65-67), rather than AUG (bases19-21)
is centered on a loop, as shown in Fig. 5. Moreover, S1nuclease
cleaves preferentially at that site just as it does at theanticodon
of tRNA. Thus there is an evident region of resem-blance of
tertiary structure of 0161 to tRNA but it does notinclude the
11-base-long region of sequence identity. The sig-nificance of the
sequence identity is entirely unexplained. Itmay indicate a
sequence recognized by the aminoacylatingenzyme, an evolutionary
vestige, or coincidence. The problemof recognition of tRNA sites by
their aminoacylating enzymesis a complex one and after a variety of
studies over a period ofmany years is only partially solved. (For a
comprehensive re-view, see ref. 30.) The corresponding problem with
chargeableviral messenger RNAs is equally formidable and may not
beworth pursuing until more is known about the functional
sig-nificance of the charging.Our sequence data for 0161 do not
provide immediate in-
sight regarding the function of the noncoding region at the
3'end of the BMV RNAs. Whatever that function may be, it
hasresulted in an evolutionary constraint that provides
nearlyidentical 0161 sequences for the four BMV RNAs even thoughthe
proteins encoded in these RNAs are different.The 0161 regions of
the BMV RNAs are not necessarily in-
volved in translation and amino acid transfer even though thisis
an important function of tRNA. Indeed, there is suggestiveevidence
to the contrary. BMV RNA that has been chemicallymodified to
preclude acceptance of tyrosine is still fully capableof serving as
a messenger in vitro (16). Certainly, pertinent invivo studies are
needed.
Possibly, the 3' ends play an important role in initiation
ofviral assembly.
Possibly the structure at the BMV RNA 3' ends is an impor-tant
feature of RNA replication. Elongation factors whosenormal function
is in translation are needed for the replicationof the RNA of phage
QB (32). Tryptophan tRNA serves as aprimer in the synthesis of Rous
sarcoma viral RNA (33). Perhapsalso, with viruses such as BMV,
structures nominally associatedwith translation have a role in
replication.We thank Christopher Sempos and Chris Saris for
technical assis-
tance, Dr. G. Altman for computer analysis, and Drs. J. E.
Dahlbergand D. Zimmern for helpful discussions. This research was
supported
by Grant CA 15613 and Training Grant T32 CA09075 from the
Na-tional Cancer Institute, Grant AI 01466 and Research Career
AwardAI 21942 from the National Institute of Allergy and Infectious
Diseases,and by Contract 1633 from the Biological Division of the
Energy Re-search and Development Administration.
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