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RNA codons and protein synthesis
J. Mol. Biol. (1966) 21, 655-570
RNA Codons and Protein Synthesist XI. Template Activity FRITZ ROTTIW$
of Modified RNA Codons
AND M&SHALL
NIRENBERQ
Laboratory of Biochemical Genetics, National Heart Institute National Institutes of Health, Bethesda, Maryland, U.S.A. (Received 22 April 1966, and in revised form 18 July 1966) Substituting 5’-, 2’-, 3’-terminal or 2’-internal ribose hydroxyls of oligonucleotides markedly affected their template activity in directing the binding of AA-sRNA§ to ribosomes. The relative template activity of oligo U preparations was as follows: p-Q’-UpUpU> UpUpU> CH,O-p-6’-UpUpU> UpUpU-3’-p> UpUpU3’-p-OCH3 >UpUpU-2’,3’-cyclic phosphate. Trimers with (2’-5’) phosphodiester linkages, (2’-5’)-UpUpU and also (2’~6’)-ApApA, did not serve as templates for phenylalanineor lyaine-sRNA, respectively. The relative template efficiency of oligo A preparations was as follows: p-5’-ApApA>ApApA>ApApA-3’-p> ApApA-S/-p. The hexamer, ApApApApApA was considerably more active as a template than the corresponding pentamer. These data indicate that two adjacent triplets are recognized by two AA-sRNA molecules bound to nearby ribosomal sites. A doublet with 5’-terminal phosphate, pUpC, served as a template for serinesRNA, whereas a doublet without terminal phosphate, UPC, did not. Although the template efficiency of pUpC was lower than that of the triplet UpCpU the data show that serine-sRNA can recognize pUpC.
1. Introduction RNA preparations with terminal substitutions have been studied as templates for both in vitro protein synthesis (Michelson & Grunberg-Manago, 1964; Abell, Rosini & Ramseur, 1965) and AA-sRNA binding to ribosomes (Nirenberg & Leder, 1964). The inhibition of polynucleotide-directed protein synthesis by oligonucleotides with terminal substitutions also has been investigated (Jones, Townsend, Sober, & Heppel, 1964; Coutsogeorgopoulos & Khorana, 1964). AA-sRNA binding studies provide a means of directly assessing the template activity of chemically defined oligonucleotides. Trinucleotides with 5’-terminal phosphate are more active as templates for AA-sRNA binding than trinucleotides without terminal phosphate, whereas trinucleotides with 2’(3’)-terminal phosphate are less active. These studies have led to the proposal that RNA and DNA contain three classes of codons, differing in structure; 5’-terminal-, 3’-terminal- and internal-codons, and t Paper X of this series appeared in J. Mol. Biol. (1966), 21, 146. 1 Present address: Department of Biochemistry, Michigan State University, Mich., U.S.A. § Abbreviationawed: AA-sRNA, aminoacyl-sRNA; DCC, dicyclohexylcarbodiimide; 5’-phosphate; Up, uridine-3’-phosphate; U2’(3’)p, mixture of uridine-2’-phosphate 3’-phosphate; (UP)~, UpUpUp; U>p, uridine-2’,3’-cyclic phosphate. 655
East
Lansing,
pU, uridine. and uridine-
666
F. ROTTMAN
AND
M. NIRENBERU
that modifications of ribose and deoxyribose hydroxyls in RNA or DNA may codon activity and hence may serve as regulatory mechanisms in protein (Nirenberg & Leder, 1964). This work was undertaken to clarify further the salient features of codon by observing the effect of substituting ribose hydroxyl groups upon the activity of RNA codons.
influence synthesis structure template
2. Materials and Methods (a) Chrornatographic and electrophoretic m&ode Oligonucleotide fractions were separated by descending paper chromatography on Whatman 3MM paper for 36 to 48 hr (fractions with terminal phosphate) or for 18 to 24 hr (fractions without terminal phosphate) with solvent A, n-propanol-concn NH,OHwater, 66 : 10 : 36 by vol. Descending paper chromatography also was performed on Whatman no. 40 paper for 7 hr with solvent B, 40 g ammonium sulfate dissolved in 100 ml. of O-1 M-sodium phosphate, pH 7.0. Chromatograms employing Whatman DE81 (DEAE) paper were developed with solvent C, O-2 ~-ammonium formate, pH 6.4, or solvent D, 0.7 M-ammonium formate, pH 6.4, for 6 hr. Separations using paper electrophoresis were performed with 0.06 M-ammonium formate (pH 2.7) at 80 v/cm using a Gilson model D electrophoresis unit. Oligonucleotide bands were located under ultraviolet light and eluted with water. (b) Oligonucleotidea prepared with micrococcal nucleate Poly U and poly A (Miles Chemical Co.) were partially degraded with micrococcal nuclease free of phosphomonoesterase activity (Worthington Biochemical) to give oligonucleotides with 3’-terminal phosphate (Alexander, Heppel & Hurwitz, 1901). In a typical experiment optimized for the production of (Up), a solution containing 100 mg of poly U adjusted to pH 8.6 with KOH was incubated with O-1 M-glycine-NaOH, pH 8.0; 0.01 MC&Cl,; and 0.16 mg protein of micrococcal nuclease in a total volume of 6.0 ml. for 4.6 hr at 37°C. The incubation mixture was frozen and lyophilized to dryness. The residue was dissolved in a small volume of water and the oligonucleotides were separated by paper chromatography on Whatman 3MM for 48 hr with solvent A. The failure to detect free nucleoside and dinucleoside monophosphate on the chromatograms indicated that the micrococcal nucleate preparation was free of phosphomonoesterase activity, Generally, the yields were as follows: 16 to 20% (Up)z, 20 to 26% (UP)~, 10 to 16% (UP)~, 10 to 16% fraction (UPL? and the remainder, Up. The relative proportion of each oligonucleotide appeared to be a function of enzyme concentration rather than time of incubation. Oligo A fractions with 3’-terminal phosphate were prepared by digestion of poly A (40 mg) in reactions containing the following components: 0.1 M-glycine-NaGH (pH 8.6); 3 x 10-e M-CaCl,; and 0.03 mg micrococcal nuclease. The reaction mixture (10 ml. total volume) was incubated at 37°C for 2 hr. The relative yields of oligo A fractions were similar to those obtained with oligo U. To prepare oligonucleotides without terminal phosphate, oligo U or oligo A fractions with 3’-terminal phosphate isolated as described above were dephosphorylated by incubation with Eschwichiu coli alkaline phosphatase. Reactions contained, in a total volume of 1 ml.: pH 8.6; 30 Azeo units of oligonucleotide with 3’-terminal phosphate; 0.06 M-(NH&CO,, and 20 pg E. coli alkaline phosphatase free of diesterase activity (Worthington, chromatographically puritled). Reaction mixtures were incubated for 3 hr at 37’C and then lyophilized to dryness. The oligonucleotides were dissolved in a minimal amount of water and separated by paper chromatography on Whatman 3MM with solvent A. It was essential to elute and rechromatograph each ultraviolet-absorbing band twice with solvent A to reduce the level of contamination by oligonucleotides of shorter chain length. After the final chromatographic step, each band was eluted and further purified by paper electrophoresis (Whatman 3MM which previously had been washed with 7% acetic acid and then extensively with water) with 0.06 r&ammonium formate, pH 2.7.
TEMPLATE
ACTIVITY
(c) Oligonucleotides
OF
MODIFIED
with
(2’-5’)
RNA
CODONS
557
diester linkages
Oligonucleotides containing a random mixture of (2’-5’) and (3’-5’) internal phosphodiester linkages were chemically synthesized by the polymerization of either uridine-2’(3’)phosphate or addenosine-2’(3’)-phosphate with diphenylphosphorochloridate by the method of Michelson (1959). Unreacted starting material and shorter oligonucleotides were removed by dialysis against water for 18 hr at 3°C. The (3’-5’) phosphodiester linkages of oligo U fractions were hydrolyzed by incubation with pancreatic RNase A. A lo-ml. reaction contained: 100mg oligo U with both (2’-5’) and (3’-5’) phosphodiester linkages and 1.1 mg pancreatic RNase A (Sigma type III-A, Sigma Chem. Co.). The pH was held at 8.0 during incubation by the addition of NH,OH. The reaction mixture was incubated at 37’C for 24 hr. The yields of (2’5’) oligonucleotides were as follows: 12 to 15% (UP)~. 4 t,o 5% (Up), and 1 to 2% (Up),. The (3’-5’) phosphodiester linkages in oligo A fractions were hydrolyzed by exhaustive incubation with T-2 RNase, generously provided by Dr George Rushizky. The reaction mixture contained the following components in a total volume of 10 ml.: 40 mg of oligo A containing a mixture of (2’-5’) and (3’-5’) phosphodiester linkages; 0.5 M-ammonium acetate, pH 4.5, and T-2 ribonuclease. Following incubation for 18 hr at 37”C, the mixtures were lyophilized. The yields of (2’-5’) oligonucleotides were: 10 to 12% (Ap),, 0 t*o 8% (APL and 2 to 3% (AP),. Oligonucleot,ides containing (2’-5’) phosphodiester linkages were separated by paper chromat,ography using solvent A for 48 hr. Ultraviolet-absorbing bands corresponding to U-2’-p-5’-U-3’-p, U-2’-p-5’-U-2’-p-5’-U-3’-p, etc., up to the pentamer, were eluted with water. Each fraction was then treated with E. coli alkaline phosphatase to remove 3’terminal phosphate as described above. After dephosphorylation, oligonucleotide fractions were further purified by paper chromatography with solvent A, elution of ultravioletabsorbing bands, and paper electrophoresis at pH 2.7 as described above. (d) Oligonucleotides prepared by RNase-catalyzed nucleoside-Z’, 3’-cyclic phosphates
addition
of
RNase A has been used to catalyze the synthesis of trinucleoside diphosphates by the addition of uridineor cytidine-2’,3’-cyclic phosphate to nucleoside or dinucleoside monophosphate acceptors (Bernfleld, 1965). The following compounds were synthesized by ribonuclease A-catalyzed addition of pU-2’,3-‘cyclic phosphate or CHsO-pU-2’,3’cyclic phosphate: pUpUpC, pUpC, and CHaO-pUpUpU. The methodology employed for the synthesis of these compounds will be described elsewhere (Bernfield & Rottman. 1966, manuscript in preparation). Briefly, the syntheses involved treatment of uridine with polyphosphoric acid followed by hydrolysis of pyrophosphate linkages to give 5’-phosphoryluridine-2’(3’)-phosphate (pU-2’(3’)p) (Hall & Khorana, 1955; Michelson, 1958). The latter compound was converted to CH,O-pU-2’,3’-cyclic phosphate by reaction with dicyclohexylcarbodiimide and excess tri-n-butylamine in methanol. Treatment of pup with aqueous ethylchloroformate in the presence of excess tri-n-butylamine produced pU-2’,3’-cyclic phosphate. UpUpC-3’-p was synthesized by the RNase A-cat’alyzed addition of U-2’,3’-cyclic phosphate to C-3/-p prepared as follows: a reaction mixture containing 0.06 ~-C-2/,3’cyclic phosphate; 0.05 M-(NH&CO,, pH 9; and 44 pg of RNase A in a total volume of 4 ml. was incubated for 32 hr at 37°C. RNase A was removed by passing the reaction mixture through a Dowex 50 (NH,+) column (2 cm x 10 cm) and elution with water. UpUpC, a gift from Dr D. Hatfield, was prepared by the RNase A-catalyzed addition of U-2’,3’-cyclic phosphate to cytidine. (e) Synthesis
of UpUpU-3’-p-OCH3
RNase A-catalyzed methanolysis of poly U in system at -20°C was employed for the preparation t Treatment
solvent Findlay,
of oligonucleotides containing 3’-terminal phosphate with DCC in methanol in the formation of phosphodiester linkages to methanol since the formation of 2’,3’ cyclic phosphate is favored (Khorana, 1959).
does not result
terminal 36*
a mixed aqueous-organic of UpUpU-3’-p-0CHs.t
558
F.
ROTTMAN
AND
M.
NIRENBERG
Math& & Rabin (1962) have shown that RNase A actively catalyzes the formation of C-3’-p-OCH, from C-2/,3’-cyclic phosphate, in reactions containing high concentrations of methanol. The conditions which we have used are as follows : 30 mg of poly U were dissolvedin0~6ml.of 0*05m-ethylenediamine-HClbuffer,pH 7.0.The additionof2.4ml. of methanol precipitated the poly U which was redissolved by the further addition of 1.8 ml. of formamide (reagent grade, Fisher). Chromatographically purified RNase A (0.25 mg Sigma type III-A dissolved in 0.12 ml. water) was added and the reaction kept at -20°C for 18 hr. The reaction flask was placed in an ice-water bath and the methanol and water were quickly removed under vacuum using an oil pump. To remove the forma. mide, the vacuum was maintained while the flask was rotated in a 70°C water bath for 5 to 10 min. It is important to remove formamide since it interferes with later chromatographic separations. The dry residue was dissolved in 6 ml. of 80% acetic acid containing 10e3 M-CuCl, to inhibit RNase activity and incubated at room temperature for 1 hr to cleave te rminal 2’,3’-phosphodiester linkages. The acetic acid was removed under vacuum and the residue dissolved in a minimal amount of water. Oligonucleotides were then purified by paper chromatography for 18 hr with solvent A. No U-2’,3-‘cyclic phosphate was observed (mobility of chromatographic standard, 27 cm) but bands corresponding to U-3’-p-OCH, (29 cm), UpU-3’-p-OCH, (21.5 cm), UpUpU-3’-p-OCH, (15 cm), UpUpUpU-3’-p-OCH, (11.5 cm), and U-3/-p (19 cm) were noted. The trimer band was eluted and further purified by paper electrophoresis at pH 2.7, as described above. (A slower migrating impurity was removed by electrophoresis.) The trimer band was eluted and again subjected to chromatography with solvent A as described above. Final yield of UpUpU-3’-p-OCH, was approximately 18 to 20%. Treatment with acetic acid to cleave terminal 2’,3’-phosphodiester linkages did not result in significant randomization of internal phosphodiester linkages, for digestion of UpUpU-3’-p-OCH, with pancreatic RNase resulted in greater than 97% hydrolysis of phosphodiester bonds. Further indication that the 3’terminal phosphodiester linkage was to methanol rather than a terminal 2’,3’-cyclic phosphate was obtained by incubation of the oligonucleotide with a 2’,3’-cyclic phosphodiesterase preparation (Anraku, 1964) obtained from E. coli (the generous gift of Dr L. Heppel) which converts U-2’,3-‘cyclic phosphate to uridine. Reactions contained the following components in a total volume of 60 ~1.: 2.7 A,,, units of either UpUpU-2’,3’-cyclic phosphate or UpUpU-3’-p-OCH, ; 5 x 10V3 M-MgCl, ; 1 x 10-3~-CoC1,; 0.06 M-sodium acetate, pH 6.0, and 20 ~1. of enzyme preparation. The reactions were incubated for 1 hr at 37’C, lyophilized to dryness, and applied to Whatman DE81 paper. Development of the chromatogram with solvent C revealed conversion of UpUpU-2’,3’-cyclic phosphate to UpUpU whereasUpUpU-3’-p-OCH,remainedunchanged. (f) Other oligonucleotides Poly U and poly A were incubated with pork liver nuclease (Heppel, personal communication) using condit.ions described previously (Nirenberg & Leder, 1964), to prepare of ApApA-2’-p and ApApA-3’-p) pUpUpU and pApApA. ApApA-2’(3’)-p (a mixture was obtained by incubating 100 mg of poly A in 20 ml. of 7-O M-NH,OH at 37’C for 24 hr (Nirenberg & Leder, 1964). ApApA-2)-p, prepared from ApApA-2’,3’-cyclic phosphate by treatment with an enzyme obtained from beef pancreas which specifically cleaves the terminal cyclic phosphodiester, was a generous gift from Drs Daniel Levin and Leon Heppel. UpUpU-2’,3’-cyclic phosphate was synthesized by treating UpUpU-3’-p with DCC and tri-n-butylamine in methanol as described by Coutsogeorgopoulos L Khorana (1964). (g) C%aracterization.
of oligonucleotidee
Threeo.~.unitsof eacholigonucleotideweresubjectedtotwo-dimensionalpa~rchro~tography (Whatman no. 40) as follows: first dimension, solvent A, 18 hr; second dimension, solvent B, 6 hr. In addition, 3 O.D. units were chromatographed on Whatman DE81 (DEAE) paper and developed for 5 hr with either solvent C or solvent D, the latter being used for oligonucleotides containing a chain-length greater than three. In the absence of any ultraviolet-absorbing contaminants, the purity of an oligonucleotide was assumed
TEMPLATE
ACTIVITY
OF
MODIFIED
RNA
CODONS
659
to be greater than 98%. Such was the case for all oligonucleotidea with the exception of the following: (Up), contained 2% (Up)s, (Up), contained 3% (Up)e, (UP)~ contained 2% (Up), and (Up), contained 1% (Up),. These contaminants were not removed by repetitive (3 times) chromatography with solvent A and electrophoresis with 0.06 M-ammonium formate at pH 2.7. Preparations of UpC and pUpC were estimated to be greater than 99% pure by subjecting 7.5 O.D. units of each (sample spot, O-5 cm diameter; doublet spot after chromatography, approximately 2 cm) to two-dimensional chromatography as stated above. Chain length and base composition of oligonucleotides were determined by digestion of 3 O.D. units with either snake venom phosphodiesterase (free of monoesterase activity), T-2 ribonuclease, or RNase A, as previously described (Nirenberg & Leder, 1964; Leder & Nirenberg, 1964; Bernfield & Nirenberg, 1965). These results are shown in Table 1 together with relative chromatographic mobilities of other oligonucleotide fractions. (h) sRNA
binding
assay
E. coli B sRNA (General Biochemicals Co.) was acylated with uniformly labeled 209 pc/pmole) or L-[14C]phenylalanine (Nuclear n-[%]lysine (Nuclear Chicago Corp., in the presence of 19 unlabeled amino acids and then Chicago Corp., 282 pc/pmole) deproteinized with phenol saturated with water as described elsewhere (Nirenberg, Matthaei & Jones, 1962). The 100,OOOg supernatant solution was prepared from E. coli W3100 as described elsewhere (Nirenberg, 1963). The sRNA accepted 33.3 and 22.1 ppThe preparation of moles of [14C]lysine or [14C]phenylalanine per Ass,, unit, respectively. E. coli W3100 ribosomes (Nirenberg, 1963), components of reaction mixtures, and nitrocellulose filter assay for ribosomal bound AA-sRNA have been described elsewhere (Nirenberg & Leder, 1964). Oligonucleotides were added to reactions with calibrated ~1. pipettes. The extinction coefficients used to calculate the concentration of oligonucleotides containing A were those reported by Singer, Heppel, Rushizky & Sober (1962) and take into account the hypochromicity exhibited by these oligonucleotides.
3. Results (a) Effect of chain length and terminal phosphate In Fig. 1 is shown the relative template activity of trinucleotides containing U with 5’- or 3’-terminal hydroxyl substitutions as a function of oligonucleotide concentration. The binding of [14C!]Phe-sRNA to ribosomes was stimulated, in order of decreasing template activity, by pUpUpU> UpUpU> CHsO-pUpUpU> UpUpUp> UpUpU-3’-p-OCH,>UpUpU-2’,3’-cyclic phosphate. The enhanced activity of pUpUpU and the decreased activity of a mixture of oligo U species, some molecules with 2’-, others with 3’-terminal phosphate, upon Phe-sRNA binding to ribosomes has been reported elsewhere (Nirenberg & Leder, 1964). The results shown here demonstrate that UpUpU-3’-p is only slightly less active than UpUpU. The addition of a methyl group to a 5’- or 3’- terminal phosphate converted a phosphomonoester to a phosphodiester linkage without the introduction of an additional nucleoside and resulted in templates with greatly reduced activity. Formation of a terminal 2’,3’-cyclic phosphodiester similarly reduced template activity. In Fig. 2(a) and (b) are shown the relation between template activity and oligo U chain length, concentration, and the nature of the end-group. (Fig. 2(a), oligo U with 5’-terminal phosphate or with no terminal phosphate; Fig. 2(b), oligo U with 3’-terminal phosphate.) As previously reported, pUpUpU is a more active template, and less is required for maximal Phe-sRNA binding to ribosomes than UpUpU (Nirenberg t Leder, 1964). A gradual increase in template activity has been reported corresponding to an increase in oligonucleotide chain length (Nirenberg & Leder, 1964) and a large increase in
(2’4’)
(2’-5’) ApA A,pA
T2 SVD SVD
C
C
B
1.0/3.1
l.Ol2.0
1~0/1*1/1~0
1*0/1*0/0~9
0.23
0.76
1.05
0.65 0.56 0.92
1.08
0.27
0.71 0.44
t Methods employed to obtain oligonucleotides were as follows: A, partial digestion of poly U or poly A with micrococcal nuclease; B, partial digestion of poly U or poly A with pork liver nuclease; C, chemical synthesis (see text); D, RNase-catalyzed addition of U>p to C or Cp; E, RNase-catalyzed addition of pU>p to C or UPC; F, RNase-catalyzed methsnolysis of poly U; G, RNase-catalyzed addition of CH,O-pU>p to UpU; H treatment of UpUpUp with DCC; I, treatment of poly A with NH,OH. $ Digestion of oligonucleotides with Ta ribonuclease (T,), snake venom phosphodiesterase (SVD), and pancreatic ribonucleaae (RNase), were performed as described in the text. $ These compounds were identified by their mobilities on paper chromatography and electrophoresis. Lack of sufficient quantities of material prevented characterization by enzymio digestion.
ApApApA
(PA), ApApA
A,pA,pAp p&dp,A
0.35
l-3/3.0/0.9
A,pA,pAp
SVD
(AP),§ ApApAp
0.41
1*0/1-0/0~9 1.2/2-o/1*2
&p&p@ &pApAp
I
A
(AP)~
[2’- or 3’-p]
SVD SVD
(AP)~
SVD
0.38 0.62
0.54
4.011.0
Ap,A
Ap,A
T, T,
A
A A
0.67
A A
APAPAPAPAPAI (AP),
AP AP AP A APAPAPAPA
0.95
3.2/1-O
APAPA
ApA
A A T2
SVD
CH,PUPUPU
UPUPU>P
1.07 0.86
1.0/1.3/o+i
0.94
U,PU,PU>P
RNfMe
G H
1.24
l.Oll.0 0.9/1.0/1.1 1.0/1.0/1.2
'=,pUp,Up,U
SVD
2*1/1-o
UP,C U,pU,pUpCHa
T2
D F
UPC UPUPUPCH,
F.
602
ROTTMAN
AND
M.
8
6
4
2
Oligonucleotide
FIG. 1. Effect
NIRENBERG
(ppmoles)
with terminal methyl diester linkages on [14C]Phe.sRNA binding to ribosomes. Reaction mixtures contained, in a volume of 50 pl., 0.1 M-‘l&s-acetate, pH ‘7.2; 0.05 M-p&%3sium acetate; 0.03 M-magnesium acetate; 1.1 A,,, units of ribosomes; oligonucleotide as indicated above; and 0.155 A,,,, unit of sRNA acylated with 4.1 ppmoles of [‘W]phenylalaninc. Incubation was for 15 min at 24%.
1
0
of oligonucleotides
I
1.0.
,
2.0
1
I
I
t
I
I
3.0
4.0
0
I.0
2.0
3.0
Oligonucleotide (a)
Fm.
2. Relation
1I
4.0
(m/lmoles) (b)
between
the template activity of oligo U preparations and the binding of L1*C]Phe-sRNA to ribosomes. The activities of pUpUpU and oligonucleotides lacking a terminal phosphate are shown in (a); those containing a 3’-terminal phosphate in (b). Reaction mixtures contained in a voIume of 60 ~1. the components described in the legend to Fig. 1; oligonucleotide as specified; and 0.155 &so unit of sRNA acylated with 4.1 ppmoles of [L4C]phenylalanine. Incubation was at 24°C for 15 min.
Phe-sRNA binding has been noted with oligo U preparations of chain length nine compared with those of chain length eight (Thach & Sundararajan, 1965). The results of Fig. 2(a) show two discrete increases in template activity; one between tri-U to tetra- or penta-U, and the second between penta- and hexa-U. It is important to note that hexa-U stimulates the binding of more Phe-sRNA to ribosomes than either
TEMPLATE
ACTIVITY
OF
MODIFIED
RNA
663
CODONS
pUpUpU or UpUpU. Approximately the same concentration (2 mpmoles) of tri-, tetra-, penta- and hexa-U are required formaximal bindingof Phe-sRNAtoribosomes. As shown in Fig. 2(b), the inhibitory effect of 3’-terminal phosphate is most evident with (Up)a. The tetra- and penta-nucleotides were considerably more active than the trinucleotide with 3’terminal phosphate. However, (Up)a had less template activity than hexa-U without terminal phosphate. Similar results were obtained with oligo A fractions and [14C]Lys-sRNA (Fig. 3(a) and (b) ). Virtually no difference in template activity between ApApA, ApApApA and ApApApApA was observed at each oligonucleotide concentration tested. However, a large increase in activity was noted with ApApApApApA. The effect of 3’-terminal phosphate is shown in Fig. 3(b). Again, 3’-terminal phosphate reduced template activity; however, the influence of 3’-terminal phosphate was not as marked as that observed with corresponding oligo U fractions. A marked difference was observed between the template activity of (AP)~ and (Ap),, the latter being a more active template. The concentration of oligo A required for maximal binding of LyssRNA was considerably less than the concentration of oligo U required for maximal binding of Phe-sRNA. 4.0
I
I
3 -tj
I
I
,APAPAPAPAPA
PA
ApApApApA=A
-L
I
0
FIG.
0.5
I
I
I.0
I.5
(a) 3. Relation between the template
I
20 0 Oligonucleotide
I
I
0.5 (mpmoles)
Ii
I.5
2.0
(b)
activity of oligo A preparations and the binding of [14C]Lys-sRNA to ribosomes. The activity of pApApA and oligonucleotides without terminal phosphate are shown in (a); those with 3’-terminal phosphate are shown in (b). Each reaction mixture contained, in a volume of 50 ~1. the components described in the legend of Fig. 1; oligonucleotides as specified; and 0.150 A 260 unit of sRNA acylated with 7.0 PLpmoles of [‘Wllysine. Incubation was for 15 min at 24°C.
The step-like increase in template activity of hexa-A, compared to the corresponding pentamers, suggests that two adjacent triplets are recognized by two molecules of AA-sRNA bound to nearby ribosomal sites. Previous studies have employed oligonucleotides composed of a mixture of 2’and 3’-terminal phosphates (Nirenberg & Leder, 1964), and in some cases such mixtures appeared to be less active than oligonucleotides with 3’-terminal phosphate
664
F.
ROTTMAN
AND
M.
NIRENBERG
only. The template activity of ApApA-2’-p is compared to that of ApApA-3’-p in Fig. 4. At each concentration tested, the trinucleotide with 2’-terminal phosphate was less active than the trinucleotide with 3’-terminal phosphate. These results demonstrate that the template activity of oligo A is affected to a greater extent by phosphate substitution at the 2’- rather than the 3’terminal hydroxyl.
Oligonucleotide
hpmoles)
FIQ. 4. Effect of oligo A preparations with either t’-terminal-, mixture of 2’- and 3’.terminal-, or 2’-terminal-phosphate on the binding of [‘%]Lys-sRNA to ribosomes. ApApA and pApApA are presented for comparison. Each 50-~1. reaction mixture contained the components specified in the legend of Fig. 1; oligonucleotide aa indicated; and 0.160 A,,, unit of sRNA aoylated with 7.0 ppmoles of [‘WIlysine. Incubation was at 24% for 15 min.
(b) Template activity of oligonu&otidaP containing (2’4’) phosphodiester linkages Earlier experiments indicated a requirement for free 2’- hydroxyl groups in oligonucleotides used to direct the binding of AA-sRNA to ribosomes (Nirenberg & Leder, 1964). In an effort further to elucidate structural requirements of RNA codons, a series of oligoribonucleotides containing (2’-5’)phosphodiester linkages were examined for template activity. Though lacking free 2’-hydroxyl groups, these oligonucleotides possess adjacent unsubstituted 3’-hydroxyls. Table 2 shows the binding of [14C]Phe- and [14C]Lys-sRNA in the presence of (2’-5’) oligonucleotides. No [14C]Phe-sRNA binding to ribosomes in response to (2’-5’)UpUpU, -UpUpUpU or -UpUpUpUpU was detected (experiment 1). In addition, when these oligonucleotides were added to reactions together with UpUpU containing the natural (3’-5’) linkages, (2’-5’)-oligo U did not inhibit [14C]Phe-sRNA binding directed by the (3’-5’) templates. (2’-5’)-UpUpU, at concentrations eight times that of (3’-5’)-UpUpU, was not inhibitory to Phe-sRNA binding. In experiment 2 of Table 2 is shown similar data for the binding of [14C]Lys-sRNA in the presence of oligonucleotides containing A. Both (2’-5’)-ApApA and -ApApApA
TEMPLATE
ACTIVITY
OF
MODIFIED
were inactive as templates and also did not inhibit somes in response to (3’-5’)-ApApA.
RNA
CODONS
565
[14C]Lys-sRNA binding to ribo-
T~BLEZ Aminoacyl-sRNA
binding to ribosomes with (2’-5’) oligonucleotides Oligonucleotide
added (mpoles)
Expt (3’-5’) oligo
(2’~5’) oligo
1 -
-
6.5 UpUpU
6.0 upupupu 4.2 UpUpUpUpU 13.3 upupu 60.0 upupu
+ +
2
-
11.1 ApApA 8.4 ApApApA 11.1 ApApA 8.4 ApApApA
6.3 UpUpU 6.3 UpUpU 6.3 UpUpU
+ +
1.5 ApApA 1.5 ApApA 1.5 ApApA
[W&A-sRNA bound to ribosomes (wmoles)
[W]Phe-sRNA 0.27 0.25 0.25 0.26 1.30 1.33 1.28 [r*C]Lys-sRNA 0.45 0.47 0.48 2.12 2.38 2.06
In experiment 1 the stimulation of [14C]Phe-sRNA binding to ribosomes was measured in the presence of oligonucleotides containing U in (2’-5’) phosphodiester linkage, (3’~5’) phosphodiester linkage, and mixtures of each oligonucleotide. Experiment 2 describes similar results obtained with [i%]Lys-sRNA and oligonucleotides containing A. Reactions contained the components described in the legend to Fig. 1. Incubation was for 15 min at 24°C.
(c) Effect of terminal phosphate on recognition of synonym phenylalunine wdons As previously reported, UpUpC is slightly more active a template in directing the binding of Phe-sRNA to ribosomes than UpUpU (Bernfleld & Nirenberg, 1965). To determine whether terminal phosphate differentially influences the template activity of synonym codons, the template activities of UpUpU, UpUpUp and pUpUpU were compared with UpUpC, UpUpCp and pUpUpC (Fig. 5). As described earlier, UpUpUp has less template activity than UpUpU. In contrast, UpUpCp and UpUpC were almost equally active as templates. 5’-Terminal phosphate enhanced the template activity of both pUpUpC and pUpUpU. However, one differential effect may deserve emphasis, i.e., 3’-terminal phosphate reduced the template activity of UpUpUp relative to UpUpU without concomitantly affecting the activity of UPUPCP. Equimolar mixtures of UpUpUp plus UpUpCp and also pUpUpU plus pUpUpC were tested for their ability to stimulate Phe-sRNA binding (Fig. 5). Oligonucleotide mixtures were assayed at both limiting and saturating concentrations. In no instance did the amount of Phe-sRNA binding directed by a mixture of synonym codons
606
F.
ROTTMAN
AND
M.
NIRENBERG
exceed that expected for an equimolar concentration of either UpUpCp or pUpUpC. These results are in accord with previous observations that one species of Phe-sRNA may recognize both UpUpU and UpUpC.
I.5 I;;/
.:&+$li$ii~
“P”PC
‘h
, 0
I
I
t
I.0
20
3.0
I
4.0 0
Oligonucleotide (a)
FIG. 6. The effect of the synonym
“PUPCP I
,
I
8
I
I
IO
20
3.00
0.2
0.4
0.6
04
(mpmoles) b)
codons, UUU and UUC, on the binding to ribosomes.
(cl
of [l%]Phe-sRNA
The activity of oligonuoleotides without terminal phosphate are shown in (a); with 3’terminal phosphate in (b); and with 5’-terminal phosphate in (c). It should be noted that the scale of the abscissa in (c) differs from the scales of (a) and (b). The symbol (0) at 1.05 mpmoles in (b) represents the amount of Phe-sRNA bound in the presence of 0.49 mpmole of UpUpUp plus 0.56 mpmole of UpUpCp. (At saturating concentrations of oligonucleotide, 5.85 mpmoles of UpUpCp directed the binding of 1.41 ppmoles of [W]Phe-sRNA while 2.50 mpmoles of UpUpUp plus 2.95 mpmoles of UpUpCp directed the binding of 1.12 ppmoles of [W]Phe-sRNA (datanot shown).) The symbol ( 0) at 0.20 mqole in (0) represents the amount of Phe-sRNA bound in the presence of 0.10 mpmole of pUpUpU plus 0.10 mpmole of pUpUpC (at saturating concentrations, 2.29 mpmoles of pUpUpC directed the binding of 1.33 ppmoles of [W]Phe-sRNA while 2.29 mpmoles of pUpUpC plus 1.77 mpmoles of pUpUpU directed the binding of 1.28 ppmoles of [‘*C]PhesRNA to ribosomes (data not shown)). Reaction mixtures contained in a volume of 50 PI., the components described in the legend of Fig. 1; oligonucleotides as shown; and 0.155 Aaso unit of sRNA aoylated with 4.1 pvoles of [Wlphenylalanine.
(cl) Effect of 5’4erminal phosphate upon the template activity of a doublet To determine whether correct recognition of two out of three bases in a codon may sometimes be sufficient to permit protein synthesis, the effect of the doublets pUpC and UpC upon the binding of [14C]Ser-sRNA to ribosomes was studied (Fig. 6(a)). The doublet with 5’4erminal phosphate, pUpC, stimulated Ser-sRNA binding to ribosomes, whereas UpC was without effect. The template activity of pUpC was proportional to the concentration within the range 0 to 60 mpmoles. To avoid contamination of pUpC with oligonucleotides of longer chain length, the doublet preparation was extensively purified before use (see Materials and Methods). No ultraviolet-absorbing contaminants were detected. The relationship between Mg2+ concentration and template activity of pUpC, UpC and UpCpU for Ser-sRNA is shown in Fig. 6(b). pUpC and UpCpU stimulated Ser-sRNA binding in reactions containing 0.02 to 0.08 M-Mg2+. The doublet UpC had no effect upon Ser-sRNA binding at each Mg2+ concentration tested. The specificity of pUpC and UpC for other [14C]-labeled AA-sRNA preparations which respond to triplets containing the sequence UpC was also studied. As shown in Table 3, pUpC stimulated the binding to ribosomes of Ser-sRNA but had no effect upon the binding of [14C]Leu-, and [14C]Ile-sRNA.
TEMPLATE
0
20
ACTIVITY
OF
40
80
60
Oligonucleotide
(mpmoles)
MODIFIED
0
RNA
CODONS
567
0.02 0.04 0.06 0.08 040 Concn McJ~+(M)
(a)
(b)
FIG. 6. The effect of UpC and pUpC on the binding of [‘%]Ser-sRNA to ribosomes. The relation between oligonucleotide concentration and [W]Ser-sRNA binding at 0.03 ar-Mg2 + is shown in (a). It should be noted that the ordinate begins at 1.25. The relation between (Mga+) concentration and [i%]Ser-sRNA binding is shown in (b). 50mpmoles of UPC, or pUpC, or 15 mpmolcs of UpCpU, were added to each reaction indicated. The remaining components of each 50-111.reaction included 0.420 Azao unit of sRNA acylated with 14.3 ppmoles of [i*C]serine plus the additions described in the legend of Fig. 1. Incubations were for 15 min at 24°C.
TABLET Effect of UpC and pUpC on AA-sRNA
SIX
Ile Val
63 mpmoles of either UpC or ponents of each 50-~1. reaction of the following AA-sRNA’s; [i4C]serine; [‘*C]Ile-sRNA, 0.34 sRNA, 0.32 A,,, unit acylated at 24°C.
None UPC PUPC None UPC PUPC None UPC PUPC
binding to ribosomes
1.45 1.53 1.98 0,22 0.31 0.25 0.34 0.29 0.31
pUpC were present in the reaction mixture. The remaining comincluded the additions described in the legend to Fig. 1 plus one [W]Ser-sRNA, 0.42 Azaa unit acylated with 14.3 pmnoles of A 2s0 unit acylated with 12.7 ppmoles of [‘%]isoleucine; [‘%]Valwith 16.8 ppmoles of [Wlvaline. Incubations were for 15 min
608
F.
ROTTMAN
AND
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NIRENBERG
These results show that a doublet with 5’-terminal phosphate, pUpC, directs Ser-sRNA binding to ribosomes. Ser-sRNA binds to ribosomes in response to triplets containing the sequence UpC in the first and second positions (UpCpU, UpCpC, UpCpA and UpCpG). UpC and pUpC did not stimulate binding of [14C]Val- or [14C]Ile-sRNA. These sRNA preparations respond to triplets containing the sequence UpC in the second and third positions. The data obtained in this study and related results obtained previously are summarized in Table 4. TABLET Relative template activity of substituted oligondeotides
OH (2’) OH (3’)
(5’)HO Template aotivityt relative to UpUpU
Oligonucleotide
p-5’-upupu UPUPU CHsO-pUpUpU upupu-3’-p UpUpUp-OCHs UpUpU-2’,3’-cyclic (2’-5’)-UpUpU Oligodeoxy T$
510 100 74 48 18 17 0 0
p
Template activity7 relative to ApApA
Oligonucleotide
p-5’-ApApA APAPA ApApA-3’-p ApApA-2*-p (2’-5’)-ApApA Oligodeoxy A$
181 100 51 15 0 0
t Relative template activities are approximations obtained by comparing the amount of AA-sRNA bound to ribosomes in the presence of limiting concentrations of oligonucleotides (0.50 mpmole of oligonucleotides containing U and 0.12 mpmole of oligonucleotides containing A) using the data presented in Figs 1, 2, 3 and 4. # Previous observations (Nirenberg & Leder, 1964).
4. Discussion Structural aspects of RNA codons which have been found to influence codon recognition include (3’~5’) phosphodiester linkages and free 2’-hydroxyl groups, since oligonucleotides with (2’~6’) phosphodiester linkages and oligonucleotides without 2’-hydroxyls (Nirenberg & Leder, 1964) have little or no template activity for AA-sRNA. Conversion of a S’-terminal phosphomonoester to a phosphodiester,
TEMPLATE
ACTIVITY
OF MODIFIED
RNA
CODONS
569
i.e. converting pUpUpU to either CHsO-pUpUpU or UpUpUpU, lowered template activity. Esterification of a terminal phosphomonoester with methanol enables one to examine the effects of adjacent phosphodiester linkages upon codon recognition without the introduction of nucleosides which might interact with ribosomes or sRNA. However, it is possible that the hydrophobic or steric character of the methyl group might influence template activity. The presence of a diester linkage on the 3’4erminus also inhibits template activity. This was observed with both UpUpUp-OCH, and UpUpU-2’,3’-cyclic phosphate. However, the latter compound represents both 2’- and 3’-terminal substitutions, and 2’- and 3’-terminal dies&s. The observed preference for 5’-terminal phosphomono- and diesters in comparison to 3’4erminal phosphomono- and dies&s may reflect a preferred polarity of interaction between the mRNA and the ribosome. Since 5’-terminal phosphate enhances template activity, it seems probable that a triphosphate attached to a 5’-terminal codon also would greatly affect template activity. RNA polymerase catalyzes the in vitro synthesis of mRNA with 5’terminal triphosphate (Maitra & Hurwitz, 1965; Bremer, Konrad, Gaines & Stem, 1965). The possibility that the 5’-terminus of mRNA specifies the attachment of the message to the ribosomes, selects the first word to be read, and thereby phases the reading, deserves to be emphasized. It should be noted that the terminal initiator codon may differ from an internal initiator codon in a polycistronic message, and similarly that a terminator codon at the end of an mRNA chain may differ from an internal terminator codon. It is possible that 5’- or 3’-terminal phosphomono- or diesters may impose restraints upon the orientation of terminal bases at ribosomal sites during codon recognition. Since one molecule of sRNA may recognize two or more synonym codons via alternate base pairing, it seems likely that terminal phosphate may differentially iniluence the template activity of synonym codons. The observation that 3’-terminal phosphate decreases the template activity of UpUpU but has little effect upon the activity of UpUpC is in accord with this suggestion. The effect of the doublet, pUpC, on Ser-sRNA binding demonstrates that certain for AA-sRNA. The effect of dinucleotides may have intrinsic template activity 5’-terminal phosphate deserves special emphasis, since UpC had no detectable template activity. Although it is clear that pUpC directs the binding of sRNA to ribosomes, we cannot distinguish between the recognition of one doublet or the overlapping recognition of three bases of adjacent doublets. It is particularly intriguing to relate the recognition of a doublet codon by sRNA to the evolution of the code. It is a pleasure to thank Miss Norma Zabriskie for her invaluable (F. R.) was supported by an American Cancer Society postdoctoral
assistance. One of us fellowship PF244.
REFERENCES Abell, C. W., Rosini, L. A. & Ramseur, M. R. (1965). Proc. Nut. Acud. Sci., Wash. 54, 608. Alexander, M., Heppel, L. A. & Hurwitz, J. (1961). J. Biol. Chem. 236, 3014. Anraku, Y. (1964). J. Biol. C?zem. 239, 3412. Bernfield, M. R. (1965). J. Biol. Chem. 240, 4753. Bernfield, M. R. & Nirenberg, M. W. (1965). Science, 147, 479. Bremer, H., Konrad, M. W., Gaines, K. & Stent, G. S. (1965). J. Mol. Biol. 13, 540. Coutsogeorgopoulos, C. & Khorana, H. G. (1964). J. Amer. Chem. Sot. 86, 2926. Findlay, D., Mathias, A. P. & Rabin, B. R. (1962). Biochmn. J. 85, 134.
F.
570 Hall,
R. H. & Khorana,
ROTTMAN
AND
H. G. (1965). J. Amer.
M.
NIRENBERG
Chem. Xoc. 77, 1871.
Jones, 0. W., Townsend, E., Sober, H. A. & Heppel, L. (1964). Biochemistry, 3, 238. Khorana, H. G. (1959). J. Amer. Chem. Sot. 81, 4657. Leder, P. & Nirenberg, M. (1964). Proc. Nat. Acrd. Sci., wash. 52, 420. Maitra, U. & Hurwitz, J. (1965). Proc. Nut. Ad. Sci., Wash. 54, 815. Michelson, A. M. (1968). J. Chem. Sot. 1957. Michelson, A. M. (1959). J. Chem. Sot. 1371. Michelson, A. M. & Grunberg-Manago, M. (1964). Biochim. biophys. A&z, 91, 92. Nirenberg, M. W. (1963). In Methods in Enzym,oZogy, ed. by S. P. Colowick & N. 0. Kaplan, vol. 6, p. 17. New York: Academic Press. Nirenberg, M. & Leder, P. (1964). Science, 145, 1399. Nirenberg, M., Matthaei, J. H. &Jones, 0. W. (1962). Proc. Nut. Acud. Sci., Wash. 48, 104. Singer, M. F., Heppel, L. A., Rushizky, G. W. & Sober, H. A. (1962). Biochim. biophys. Acta, 61, 474. Thach, R. E. & Sundaritrajan, T. A. (1965). Proc. Nat. Acud. Sci., Wash. 53, 1021.
RNA Codons and Protein Synthesist XI. Template Activity FRITZ ROTTIW$
of Modified RNA Codons
AND M&SHALL
NIRENBERQ
Laboratory of Biochemical Genetics, National Heart Institute National Institutes of Health, Bethesda, Maryland, U.S.A. (Received 22 April 1966, and in revised form 18 July 1966) Substituting 5’-, 2’-, 3’-terminal or 2’-internal ribose hydroxyls of oligonucleotides markedly affected their template activity in directing the binding of AA-sRNA§ to ribosomes. The relative template activity of oligo U preparations was as follows: p-Q’-UpUpU> UpUpU> CH,O-p-6’-UpUpU> UpUpU-3’-p> UpUpU3’-p-OCH3 >UpUpU-2’,3’-cyclic phosphate. Trimers with (2’-5’) phosphodiester linkages, (2’-5’)-UpUpU and also (2’~6’)-ApApA, did not serve as templates for phenylalanineor lyaine-sRNA, respectively. The relative template efficiency of oligo A preparations was as follows: p-5’-ApApA>ApApA>ApApA-3’-p> ApApA-S/-p. The hexamer, ApApApApApA was considerably more active as a template than the corresponding pentamer. These data indicate that two adjacent triplets are recognized by two AA-sRNA molecules bound to nearby ribosomal sites. A doublet with 5’-terminal phosphate, pUpC, served as a template for serinesRNA, whereas a doublet without terminal phosphate, UPC, did not. Although the template efficiency of pUpC was lower than that of the triplet UpCpU the data show that serine-sRNA can recognize pUpC.
1. Introduction RNA preparations with terminal substitutions have been studied as templates for both in vitro protein synthesis (Michelson & Grunberg-Manago, 1964; Abell, Rosini & Ramseur, 1965) and AA-sRNA binding to ribosomes (Nirenberg & Leder, 1964). The inhibition of polynucleotide-directed protein synthesis by oligonucleotides with terminal substitutions also has been investigated (Jones, Townsend, Sober, & Heppel, 1964; Coutsogeorgopoulos & Khorana, 1964). AA-sRNA binding studies provide a means of directly assessing the template activity of chemically defined oligonucleotides. Trinucleotides with 5’-terminal phosphate are more active as templates for AA-sRNA binding than trinucleotides without terminal phosphate, whereas trinucleotides with 2’(3’)-terminal phosphate are less active. These studies have led to the proposal that RNA and DNA contain three classes of codons, differing in structure; 5’-terminal-, 3’-terminal- and internal-codons, and t Paper X of this series appeared in J. Mol. Biol. (1966), 21, 146. 1 Present address: Department of Biochemistry, Michigan State University, Mich., U.S.A. § Abbreviationawed: AA-sRNA, aminoacyl-sRNA; DCC, dicyclohexylcarbodiimide; 5’-phosphate; Up, uridine-3’-phosphate; U2’(3’)p, mixture of uridine-2’-phosphate 3’-phosphate; (UP)~, UpUpUp; U>p, uridine-2’,3’-cyclic phosphate. 655
East
Lansing,
pU, uridine. and uridine-
666
F. ROTTMAN
AND
M. NIRENBERU
that modifications of ribose and deoxyribose hydroxyls in RNA or DNA may codon activity and hence may serve as regulatory mechanisms in protein (Nirenberg & Leder, 1964). This work was undertaken to clarify further the salient features of codon by observing the effect of substituting ribose hydroxyl groups upon the activity of RNA codons.
influence synthesis structure template
2. Materials and Methods (a) Chrornatographic and electrophoretic m&ode Oligonucleotide fractions were separated by descending paper chromatography on Whatman 3MM paper for 36 to 48 hr (fractions with terminal phosphate) or for 18 to 24 hr (fractions without terminal phosphate) with solvent A, n-propanol-concn NH,OHwater, 66 : 10 : 36 by vol. Descending paper chromatography also was performed on Whatman no. 40 paper for 7 hr with solvent B, 40 g ammonium sulfate dissolved in 100 ml. of O-1 M-sodium phosphate, pH 7.0. Chromatograms employing Whatman DE81 (DEAE) paper were developed with solvent C, O-2 ~-ammonium formate, pH 6.4, or solvent D, 0.7 M-ammonium formate, pH 6.4, for 6 hr. Separations using paper electrophoresis were performed with 0.06 M-ammonium formate (pH 2.7) at 80 v/cm using a Gilson model D electrophoresis unit. Oligonucleotide bands were located under ultraviolet light and eluted with water. (b) Oligonucleotidea prepared with micrococcal nucleate Poly U and poly A (Miles Chemical Co.) were partially degraded with micrococcal nuclease free of phosphomonoesterase activity (Worthington Biochemical) to give oligonucleotides with 3’-terminal phosphate (Alexander, Heppel & Hurwitz, 1901). In a typical experiment optimized for the production of (Up), a solution containing 100 mg of poly U adjusted to pH 8.6 with KOH was incubated with O-1 M-glycine-NaOH, pH 8.0; 0.01 MC&Cl,; and 0.16 mg protein of micrococcal nuclease in a total volume of 6.0 ml. for 4.6 hr at 37°C. The incubation mixture was frozen and lyophilized to dryness. The residue was dissolved in a small volume of water and the oligonucleotides were separated by paper chromatography on Whatman 3MM for 48 hr with solvent A. The failure to detect free nucleoside and dinucleoside monophosphate on the chromatograms indicated that the micrococcal nucleate preparation was free of phosphomonoesterase activity, Generally, the yields were as follows: 16 to 20% (Up)z, 20 to 26% (UP)~, 10 to 16% (UP)~, 10 to 16% fraction (UPL? and the remainder, Up. The relative proportion of each oligonucleotide appeared to be a function of enzyme concentration rather than time of incubation. Oligo A fractions with 3’-terminal phosphate were prepared by digestion of poly A (40 mg) in reactions containing the following components: 0.1 M-glycine-NaGH (pH 8.6); 3 x 10-e M-CaCl,; and 0.03 mg micrococcal nuclease. The reaction mixture (10 ml. total volume) was incubated at 37°C for 2 hr. The relative yields of oligo A fractions were similar to those obtained with oligo U. To prepare oligonucleotides without terminal phosphate, oligo U or oligo A fractions with 3’-terminal phosphate isolated as described above were dephosphorylated by incubation with Eschwichiu coli alkaline phosphatase. Reactions contained, in a total volume of 1 ml.: pH 8.6; 30 Azeo units of oligonucleotide with 3’-terminal phosphate; 0.06 M-(NH&CO,, and 20 pg E. coli alkaline phosphatase free of diesterase activity (Worthington, chromatographically puritled). Reaction mixtures were incubated for 3 hr at 37’C and then lyophilized to dryness. The oligonucleotides were dissolved in a minimal amount of water and separated by paper chromatography on Whatman 3MM with solvent A. It was essential to elute and rechromatograph each ultraviolet-absorbing band twice with solvent A to reduce the level of contamination by oligonucleotides of shorter chain length. After the final chromatographic step, each band was eluted and further purified by paper electrophoresis (Whatman 3MM which previously had been washed with 7% acetic acid and then extensively with water) with 0.06 r&ammonium formate, pH 2.7.
TEMPLATE
ACTIVITY
(c) Oligonucleotides
OF
MODIFIED
with
(2’-5’)
RNA
CODONS
557
diester linkages
Oligonucleotides containing a random mixture of (2’-5’) and (3’-5’) internal phosphodiester linkages were chemically synthesized by the polymerization of either uridine-2’(3’)phosphate or addenosine-2’(3’)-phosphate with diphenylphosphorochloridate by the method of Michelson (1959). Unreacted starting material and shorter oligonucleotides were removed by dialysis against water for 18 hr at 3°C. The (3’-5’) phosphodiester linkages of oligo U fractions were hydrolyzed by incubation with pancreatic RNase A. A lo-ml. reaction contained: 100mg oligo U with both (2’-5’) and (3’-5’) phosphodiester linkages and 1.1 mg pancreatic RNase A (Sigma type III-A, Sigma Chem. Co.). The pH was held at 8.0 during incubation by the addition of NH,OH. The reaction mixture was incubated at 37’C for 24 hr. The yields of (2’5’) oligonucleotides were as follows: 12 to 15% (UP)~. 4 t,o 5% (Up), and 1 to 2% (Up),. The (3’-5’) phosphodiester linkages in oligo A fractions were hydrolyzed by exhaustive incubation with T-2 RNase, generously provided by Dr George Rushizky. The reaction mixture contained the following components in a total volume of 10 ml.: 40 mg of oligo A containing a mixture of (2’-5’) and (3’-5’) phosphodiester linkages; 0.5 M-ammonium acetate, pH 4.5, and T-2 ribonuclease. Following incubation for 18 hr at 37”C, the mixtures were lyophilized. The yields of (2’-5’) oligonucleotides were: 10 to 12% (Ap),, 0 t*o 8% (APL and 2 to 3% (AP),. Oligonucleot,ides containing (2’-5’) phosphodiester linkages were separated by paper chromat,ography using solvent A for 48 hr. Ultraviolet-absorbing bands corresponding to U-2’-p-5’-U-3’-p, U-2’-p-5’-U-2’-p-5’-U-3’-p, etc., up to the pentamer, were eluted with water. Each fraction was then treated with E. coli alkaline phosphatase to remove 3’terminal phosphate as described above. After dephosphorylation, oligonucleotide fractions were further purified by paper chromatography with solvent A, elution of ultravioletabsorbing bands, and paper electrophoresis at pH 2.7 as described above. (d) Oligonucleotides prepared by RNase-catalyzed nucleoside-Z’, 3’-cyclic phosphates
addition
of
RNase A has been used to catalyze the synthesis of trinucleoside diphosphates by the addition of uridineor cytidine-2’,3’-cyclic phosphate to nucleoside or dinucleoside monophosphate acceptors (Bernfleld, 1965). The following compounds were synthesized by ribonuclease A-catalyzed addition of pU-2’,3-‘cyclic phosphate or CHsO-pU-2’,3’cyclic phosphate: pUpUpC, pUpC, and CHaO-pUpUpU. The methodology employed for the synthesis of these compounds will be described elsewhere (Bernfield & Rottman. 1966, manuscript in preparation). Briefly, the syntheses involved treatment of uridine with polyphosphoric acid followed by hydrolysis of pyrophosphate linkages to give 5’-phosphoryluridine-2’(3’)-phosphate (pU-2’(3’)p) (Hall & Khorana, 1955; Michelson, 1958). The latter compound was converted to CH,O-pU-2’,3’-cyclic phosphate by reaction with dicyclohexylcarbodiimide and excess tri-n-butylamine in methanol. Treatment of pup with aqueous ethylchloroformate in the presence of excess tri-n-butylamine produced pU-2’,3’-cyclic phosphate. UpUpC-3’-p was synthesized by the RNase A-cat’alyzed addition of U-2’,3’-cyclic phosphate to C-3/-p prepared as follows: a reaction mixture containing 0.06 ~-C-2/,3’cyclic phosphate; 0.05 M-(NH&CO,, pH 9; and 44 pg of RNase A in a total volume of 4 ml. was incubated for 32 hr at 37°C. RNase A was removed by passing the reaction mixture through a Dowex 50 (NH,+) column (2 cm x 10 cm) and elution with water. UpUpC, a gift from Dr D. Hatfield, was prepared by the RNase A-catalyzed addition of U-2’,3’-cyclic phosphate to cytidine. (e) Synthesis
of UpUpU-3’-p-OCH3
RNase A-catalyzed methanolysis of poly U in system at -20°C was employed for the preparation t Treatment
solvent Findlay,
of oligonucleotides containing 3’-terminal phosphate with DCC in methanol in the formation of phosphodiester linkages to methanol since the formation of 2’,3’ cyclic phosphate is favored (Khorana, 1959).
does not result
terminal 36*
a mixed aqueous-organic of UpUpU-3’-p-0CHs.t
558
F.
ROTTMAN
AND
M.
NIRENBERG
Math& & Rabin (1962) have shown that RNase A actively catalyzes the formation of C-3’-p-OCH, from C-2/,3’-cyclic phosphate, in reactions containing high concentrations of methanol. The conditions which we have used are as follows : 30 mg of poly U were dissolvedin0~6ml.of 0*05m-ethylenediamine-HClbuffer,pH 7.0.The additionof2.4ml. of methanol precipitated the poly U which was redissolved by the further addition of 1.8 ml. of formamide (reagent grade, Fisher). Chromatographically purified RNase A (0.25 mg Sigma type III-A dissolved in 0.12 ml. water) was added and the reaction kept at -20°C for 18 hr. The reaction flask was placed in an ice-water bath and the methanol and water were quickly removed under vacuum using an oil pump. To remove the forma. mide, the vacuum was maintained while the flask was rotated in a 70°C water bath for 5 to 10 min. It is important to remove formamide since it interferes with later chromatographic separations. The dry residue was dissolved in 6 ml. of 80% acetic acid containing 10e3 M-CuCl, to inhibit RNase activity and incubated at room temperature for 1 hr to cleave te rminal 2’,3’-phosphodiester linkages. The acetic acid was removed under vacuum and the residue dissolved in a minimal amount of water. Oligonucleotides were then purified by paper chromatography for 18 hr with solvent A. No U-2’,3-‘cyclic phosphate was observed (mobility of chromatographic standard, 27 cm) but bands corresponding to U-3’-p-OCH, (29 cm), UpU-3’-p-OCH, (21.5 cm), UpUpU-3’-p-OCH, (15 cm), UpUpUpU-3’-p-OCH, (11.5 cm), and U-3/-p (19 cm) were noted. The trimer band was eluted and further purified by paper electrophoresis at pH 2.7, as described above. (A slower migrating impurity was removed by electrophoresis.) The trimer band was eluted and again subjected to chromatography with solvent A as described above. Final yield of UpUpU-3’-p-OCH, was approximately 18 to 20%. Treatment with acetic acid to cleave terminal 2’,3’-phosphodiester linkages did not result in significant randomization of internal phosphodiester linkages, for digestion of UpUpU-3’-p-OCH, with pancreatic RNase resulted in greater than 97% hydrolysis of phosphodiester bonds. Further indication that the 3’terminal phosphodiester linkage was to methanol rather than a terminal 2’,3’-cyclic phosphate was obtained by incubation of the oligonucleotide with a 2’,3’-cyclic phosphodiesterase preparation (Anraku, 1964) obtained from E. coli (the generous gift of Dr L. Heppel) which converts U-2’,3-‘cyclic phosphate to uridine. Reactions contained the following components in a total volume of 60 ~1.: 2.7 A,,, units of either UpUpU-2’,3’-cyclic phosphate or UpUpU-3’-p-OCH, ; 5 x 10V3 M-MgCl, ; 1 x 10-3~-CoC1,; 0.06 M-sodium acetate, pH 6.0, and 20 ~1. of enzyme preparation. The reactions were incubated for 1 hr at 37’C, lyophilized to dryness, and applied to Whatman DE81 paper. Development of the chromatogram with solvent C revealed conversion of UpUpU-2’,3’-cyclic phosphate to UpUpU whereasUpUpU-3’-p-OCH,remainedunchanged. (f) Other oligonucleotides Poly U and poly A were incubated with pork liver nuclease (Heppel, personal communication) using condit.ions described previously (Nirenberg & Leder, 1964), to prepare of ApApA-2’-p and ApApA-3’-p) pUpUpU and pApApA. ApApA-2’(3’)-p (a mixture was obtained by incubating 100 mg of poly A in 20 ml. of 7-O M-NH,OH at 37’C for 24 hr (Nirenberg & Leder, 1964). ApApA-2)-p, prepared from ApApA-2’,3’-cyclic phosphate by treatment with an enzyme obtained from beef pancreas which specifically cleaves the terminal cyclic phosphodiester, was a generous gift from Drs Daniel Levin and Leon Heppel. UpUpU-2’,3’-cyclic phosphate was synthesized by treating UpUpU-3’-p with DCC and tri-n-butylamine in methanol as described by Coutsogeorgopoulos L Khorana (1964). (g) C%aracterization.
of oligonucleotidee
Threeo.~.unitsof eacholigonucleotideweresubjectedtotwo-dimensionalpa~rchro~tography (Whatman no. 40) as follows: first dimension, solvent A, 18 hr; second dimension, solvent B, 6 hr. In addition, 3 O.D. units were chromatographed on Whatman DE81 (DEAE) paper and developed for 5 hr with either solvent C or solvent D, the latter being used for oligonucleotides containing a chain-length greater than three. In the absence of any ultraviolet-absorbing contaminants, the purity of an oligonucleotide was assumed
TEMPLATE
ACTIVITY
OF
MODIFIED
RNA
CODONS
659
to be greater than 98%. Such was the case for all oligonucleotidea with the exception of the following: (Up), contained 2% (Up)s, (Up), contained 3% (Up)e, (UP)~ contained 2% (Up), and (Up), contained 1% (Up),. These contaminants were not removed by repetitive (3 times) chromatography with solvent A and electrophoresis with 0.06 M-ammonium formate at pH 2.7. Preparations of UpC and pUpC were estimated to be greater than 99% pure by subjecting 7.5 O.D. units of each (sample spot, O-5 cm diameter; doublet spot after chromatography, approximately 2 cm) to two-dimensional chromatography as stated above. Chain length and base composition of oligonucleotides were determined by digestion of 3 O.D. units with either snake venom phosphodiesterase (free of monoesterase activity), T-2 ribonuclease, or RNase A, as previously described (Nirenberg & Leder, 1964; Leder & Nirenberg, 1964; Bernfield & Nirenberg, 1965). These results are shown in Table 1 together with relative chromatographic mobilities of other oligonucleotide fractions. (h) sRNA
binding
assay
E. coli B sRNA (General Biochemicals Co.) was acylated with uniformly labeled 209 pc/pmole) or L-[14C]phenylalanine (Nuclear n-[%]lysine (Nuclear Chicago Corp., in the presence of 19 unlabeled amino acids and then Chicago Corp., 282 pc/pmole) deproteinized with phenol saturated with water as described elsewhere (Nirenberg, Matthaei & Jones, 1962). The 100,OOOg supernatant solution was prepared from E. coli W3100 as described elsewhere (Nirenberg, 1963). The sRNA accepted 33.3 and 22.1 ppThe preparation of moles of [14C]lysine or [14C]phenylalanine per Ass,, unit, respectively. E. coli W3100 ribosomes (Nirenberg, 1963), components of reaction mixtures, and nitrocellulose filter assay for ribosomal bound AA-sRNA have been described elsewhere (Nirenberg & Leder, 1964). Oligonucleotides were added to reactions with calibrated ~1. pipettes. The extinction coefficients used to calculate the concentration of oligonucleotides containing A were those reported by Singer, Heppel, Rushizky & Sober (1962) and take into account the hypochromicity exhibited by these oligonucleotides.
3. Results (a) Effect of chain length and terminal phosphate In Fig. 1 is shown the relative template activity of trinucleotides containing U with 5’- or 3’-terminal hydroxyl substitutions as a function of oligonucleotide concentration. The binding of [14C!]Phe-sRNA to ribosomes was stimulated, in order of decreasing template activity, by pUpUpU> UpUpU> CHsO-pUpUpU> UpUpUp> UpUpU-3’-p-OCH,>UpUpU-2’,3’-cyclic phosphate. The enhanced activity of pUpUpU and the decreased activity of a mixture of oligo U species, some molecules with 2’-, others with 3’-terminal phosphate, upon Phe-sRNA binding to ribosomes has been reported elsewhere (Nirenberg & Leder, 1964). The results shown here demonstrate that UpUpU-3’-p is only slightly less active than UpUpU. The addition of a methyl group to a 5’- or 3’- terminal phosphate converted a phosphomonoester to a phosphodiester linkage without the introduction of an additional nucleoside and resulted in templates with greatly reduced activity. Formation of a terminal 2’,3’-cyclic phosphodiester similarly reduced template activity. In Fig. 2(a) and (b) are shown the relation between template activity and oligo U chain length, concentration, and the nature of the end-group. (Fig. 2(a), oligo U with 5’-terminal phosphate or with no terminal phosphate; Fig. 2(b), oligo U with 3’-terminal phosphate.) As previously reported, pUpUpU is a more active template, and less is required for maximal Phe-sRNA binding to ribosomes than UpUpU (Nirenberg t Leder, 1964). A gradual increase in template activity has been reported corresponding to an increase in oligonucleotide chain length (Nirenberg & Leder, 1964) and a large increase in
(2’4’)
(2’-5’) ApA A,pA
T2 SVD SVD
C
C
B
1.0/3.1
l.Ol2.0
1~0/1*1/1~0
1*0/1*0/0~9
0.23
0.76
1.05
0.65 0.56 0.92
1.08
0.27
0.71 0.44
t Methods employed to obtain oligonucleotides were as follows: A, partial digestion of poly U or poly A with micrococcal nuclease; B, partial digestion of poly U or poly A with pork liver nuclease; C, chemical synthesis (see text); D, RNase-catalyzed addition of U>p to C or Cp; E, RNase-catalyzed addition of pU>p to C or UPC; F, RNase-catalyzed methsnolysis of poly U; G, RNase-catalyzed addition of CH,O-pU>p to UpU; H treatment of UpUpUp with DCC; I, treatment of poly A with NH,OH. $ Digestion of oligonucleotides with Ta ribonuclease (T,), snake venom phosphodiesterase (SVD), and pancreatic ribonucleaae (RNase), were performed as described in the text. $ These compounds were identified by their mobilities on paper chromatography and electrophoresis. Lack of sufficient quantities of material prevented characterization by enzymio digestion.
ApApApA
(PA), ApApA
A,pA,pAp p&dp,A
0.35
l-3/3.0/0.9
A,pA,pAp
SVD
(AP),§ ApApAp
0.41
1*0/1-0/0~9 1.2/2-o/1*2
&p&p@ &pApAp
I
A
(AP)~
[2’- or 3’-p]
SVD SVD
(AP)~
SVD
0.38 0.62
0.54
4.011.0
Ap,A
Ap,A
T, T,
A
A A
0.67
A A
APAPAPAPAPAI (AP),
AP AP AP A APAPAPAPA
0.95
3.2/1-O
APAPA
ApA
A A T2
SVD
CH,PUPUPU
UPUPU>P
1.07 0.86
1.0/1.3/o+i
0.94
U,PU,PU>P
RNfMe
G H
1.24
l.Oll.0 0.9/1.0/1.1 1.0/1.0/1.2
'=,pUp,Up,U
SVD
2*1/1-o
UP,C U,pU,pUpCHa
T2
D F
UPC UPUPUPCH,
F.
602
ROTTMAN
AND
M.
8
6
4
2
Oligonucleotide
FIG. 1. Effect
NIRENBERG
(ppmoles)
with terminal methyl diester linkages on [14C]Phe.sRNA binding to ribosomes. Reaction mixtures contained, in a volume of 50 pl., 0.1 M-‘l&s-acetate, pH ‘7.2; 0.05 M-p&%3sium acetate; 0.03 M-magnesium acetate; 1.1 A,,, units of ribosomes; oligonucleotide as indicated above; and 0.155 A,,,, unit of sRNA acylated with 4.1 ppmoles of [‘W]phenylalaninc. Incubation was for 15 min at 24%.
1
0
of oligonucleotides
I
1.0.
,
2.0
1
I
I
t
I
I
3.0
4.0
0
I.0
2.0
3.0
Oligonucleotide (a)
Fm.
2. Relation
1I
4.0
(m/lmoles) (b)
between
the template activity of oligo U preparations and the binding of L1*C]Phe-sRNA to ribosomes. The activities of pUpUpU and oligonucleotides lacking a terminal phosphate are shown in (a); those containing a 3’-terminal phosphate in (b). Reaction mixtures contained in a voIume of 60 ~1. the components described in the legend to Fig. 1; oligonucleotide as specified; and 0.155 &so unit of sRNA acylated with 4.1 ppmoles of [L4C]phenylalanine. Incubation was at 24°C for 15 min.
Phe-sRNA binding has been noted with oligo U preparations of chain length nine compared with those of chain length eight (Thach & Sundararajan, 1965). The results of Fig. 2(a) show two discrete increases in template activity; one between tri-U to tetra- or penta-U, and the second between penta- and hexa-U. It is important to note that hexa-U stimulates the binding of more Phe-sRNA to ribosomes than either
TEMPLATE
ACTIVITY
OF
MODIFIED
RNA
663
CODONS
pUpUpU or UpUpU. Approximately the same concentration (2 mpmoles) of tri-, tetra-, penta- and hexa-U are required formaximal bindingof Phe-sRNAtoribosomes. As shown in Fig. 2(b), the inhibitory effect of 3’-terminal phosphate is most evident with (Up)a. The tetra- and penta-nucleotides were considerably more active than the trinucleotide with 3’terminal phosphate. However, (Up)a had less template activity than hexa-U without terminal phosphate. Similar results were obtained with oligo A fractions and [14C]Lys-sRNA (Fig. 3(a) and (b) ). Virtually no difference in template activity between ApApA, ApApApA and ApApApApA was observed at each oligonucleotide concentration tested. However, a large increase in activity was noted with ApApApApApA. The effect of 3’-terminal phosphate is shown in Fig. 3(b). Again, 3’-terminal phosphate reduced template activity; however, the influence of 3’-terminal phosphate was not as marked as that observed with corresponding oligo U fractions. A marked difference was observed between the template activity of (AP)~ and (Ap),, the latter being a more active template. The concentration of oligo A required for maximal binding of LyssRNA was considerably less than the concentration of oligo U required for maximal binding of Phe-sRNA. 4.0
I
I
3 -tj
I
I
,APAPAPAPAPA
PA
ApApApApA=A
-L
I
0
FIG.
0.5
I
I
I.0
I.5
(a) 3. Relation between the template
I
20 0 Oligonucleotide
I
I
0.5 (mpmoles)
Ii
I.5
2.0
(b)
activity of oligo A preparations and the binding of [14C]Lys-sRNA to ribosomes. The activity of pApApA and oligonucleotides without terminal phosphate are shown in (a); those with 3’-terminal phosphate are shown in (b). Each reaction mixture contained, in a volume of 50 ~1. the components described in the legend of Fig. 1; oligonucleotides as specified; and 0.150 A 260 unit of sRNA acylated with 7.0 PLpmoles of [‘Wllysine. Incubation was for 15 min at 24°C.
The step-like increase in template activity of hexa-A, compared to the corresponding pentamers, suggests that two adjacent triplets are recognized by two molecules of AA-sRNA bound to nearby ribosomal sites. Previous studies have employed oligonucleotides composed of a mixture of 2’and 3’-terminal phosphates (Nirenberg & Leder, 1964), and in some cases such mixtures appeared to be less active than oligonucleotides with 3’-terminal phosphate
664
F.
ROTTMAN
AND
M.
NIRENBERG
only. The template activity of ApApA-2’-p is compared to that of ApApA-3’-p in Fig. 4. At each concentration tested, the trinucleotide with 2’-terminal phosphate was less active than the trinucleotide with 3’-terminal phosphate. These results demonstrate that the template activity of oligo A is affected to a greater extent by phosphate substitution at the 2’- rather than the 3’terminal hydroxyl.
Oligonucleotide
hpmoles)
FIQ. 4. Effect of oligo A preparations with either t’-terminal-, mixture of 2’- and 3’.terminal-, or 2’-terminal-phosphate on the binding of [‘%]Lys-sRNA to ribosomes. ApApA and pApApA are presented for comparison. Each 50-~1. reaction mixture contained the components specified in the legend of Fig. 1; oligonucleotide aa indicated; and 0.160 A,,, unit of sRNA aoylated with 7.0 ppmoles of [‘WIlysine. Incubation was at 24% for 15 min.
(b) Template activity of oligonu&otidaP containing (2’4’) phosphodiester linkages Earlier experiments indicated a requirement for free 2’- hydroxyl groups in oligonucleotides used to direct the binding of AA-sRNA to ribosomes (Nirenberg & Leder, 1964). In an effort further to elucidate structural requirements of RNA codons, a series of oligoribonucleotides containing (2’-5’)phosphodiester linkages were examined for template activity. Though lacking free 2’-hydroxyl groups, these oligonucleotides possess adjacent unsubstituted 3’-hydroxyls. Table 2 shows the binding of [14C]Phe- and [14C]Lys-sRNA in the presence of (2’-5’) oligonucleotides. No [14C]Phe-sRNA binding to ribosomes in response to (2’-5’)UpUpU, -UpUpUpU or -UpUpUpUpU was detected (experiment 1). In addition, when these oligonucleotides were added to reactions together with UpUpU containing the natural (3’-5’) linkages, (2’-5’)-oligo U did not inhibit [14C]Phe-sRNA binding directed by the (3’-5’) templates. (2’-5’)-UpUpU, at concentrations eight times that of (3’-5’)-UpUpU, was not inhibitory to Phe-sRNA binding. In experiment 2 of Table 2 is shown similar data for the binding of [14C]Lys-sRNA in the presence of oligonucleotides containing A. Both (2’-5’)-ApApA and -ApApApA
TEMPLATE
ACTIVITY
OF
MODIFIED
were inactive as templates and also did not inhibit somes in response to (3’-5’)-ApApA.
RNA
CODONS
565
[14C]Lys-sRNA binding to ribo-
T~BLEZ Aminoacyl-sRNA
binding to ribosomes with (2’-5’) oligonucleotides Oligonucleotide
added (mpoles)
Expt (3’-5’) oligo
(2’~5’) oligo
1 -
-
6.5 UpUpU
6.0 upupupu 4.2 UpUpUpUpU 13.3 upupu 60.0 upupu
+ +
2
-
11.1 ApApA 8.4 ApApApA 11.1 ApApA 8.4 ApApApA
6.3 UpUpU 6.3 UpUpU 6.3 UpUpU
+ +
1.5 ApApA 1.5 ApApA 1.5 ApApA
[W&A-sRNA bound to ribosomes (wmoles)
[W]Phe-sRNA 0.27 0.25 0.25 0.26 1.30 1.33 1.28 [r*C]Lys-sRNA 0.45 0.47 0.48 2.12 2.38 2.06
In experiment 1 the stimulation of [14C]Phe-sRNA binding to ribosomes was measured in the presence of oligonucleotides containing U in (2’-5’) phosphodiester linkage, (3’~5’) phosphodiester linkage, and mixtures of each oligonucleotide. Experiment 2 describes similar results obtained with [i%]Lys-sRNA and oligonucleotides containing A. Reactions contained the components described in the legend to Fig. 1. Incubation was for 15 min at 24°C.
(c) Effect of terminal phosphate on recognition of synonym phenylalunine wdons As previously reported, UpUpC is slightly more active a template in directing the binding of Phe-sRNA to ribosomes than UpUpU (Bernfleld & Nirenberg, 1965). To determine whether terminal phosphate differentially influences the template activity of synonym codons, the template activities of UpUpU, UpUpUp and pUpUpU were compared with UpUpC, UpUpCp and pUpUpC (Fig. 5). As described earlier, UpUpUp has less template activity than UpUpU. In contrast, UpUpCp and UpUpC were almost equally active as templates. 5’-Terminal phosphate enhanced the template activity of both pUpUpC and pUpUpU. However, one differential effect may deserve emphasis, i.e., 3’-terminal phosphate reduced the template activity of UpUpUp relative to UpUpU without concomitantly affecting the activity of UPUPCP. Equimolar mixtures of UpUpUp plus UpUpCp and also pUpUpU plus pUpUpC were tested for their ability to stimulate Phe-sRNA binding (Fig. 5). Oligonucleotide mixtures were assayed at both limiting and saturating concentrations. In no instance did the amount of Phe-sRNA binding directed by a mixture of synonym codons
606
F.
ROTTMAN
AND
M.
NIRENBERG
exceed that expected for an equimolar concentration of either UpUpCp or pUpUpC. These results are in accord with previous observations that one species of Phe-sRNA may recognize both UpUpU and UpUpC.
I.5 I;;/
.:&+$li$ii~
“P”PC
‘h
, 0
I
I
t
I.0
20
3.0
I
4.0 0
Oligonucleotide (a)
FIG. 6. The effect of the synonym
“PUPCP I
,
I
8
I
I
IO
20
3.00
0.2
0.4
0.6
04
(mpmoles) b)
codons, UUU and UUC, on the binding to ribosomes.
(cl
of [l%]Phe-sRNA
The activity of oligonuoleotides without terminal phosphate are shown in (a); with 3’terminal phosphate in (b); and with 5’-terminal phosphate in (c). It should be noted that the scale of the abscissa in (c) differs from the scales of (a) and (b). The symbol (0) at 1.05 mpmoles in (b) represents the amount of Phe-sRNA bound in the presence of 0.49 mpmole of UpUpUp plus 0.56 mpmole of UpUpCp. (At saturating concentrations of oligonucleotide, 5.85 mpmoles of UpUpCp directed the binding of 1.41 ppmoles of [W]Phe-sRNA while 2.50 mpmoles of UpUpUp plus 2.95 mpmoles of UpUpCp directed the binding of 1.12 ppmoles of [W]Phe-sRNA (datanot shown).) The symbol ( 0) at 0.20 mqole in (0) represents the amount of Phe-sRNA bound in the presence of 0.10 mpmole of pUpUpU plus 0.10 mpmole of pUpUpC (at saturating concentrations, 2.29 mpmoles of pUpUpC directed the binding of 1.33 ppmoles of [W]Phe-sRNA while 2.29 mpmoles of pUpUpC plus 1.77 mpmoles of pUpUpU directed the binding of 1.28 ppmoles of [‘*C]PhesRNA to ribosomes (data not shown)). Reaction mixtures contained in a volume of 50 PI., the components described in the legend of Fig. 1; oligonucleotides as shown; and 0.155 Aaso unit of sRNA aoylated with 4.1 pvoles of [Wlphenylalanine.
(cl) Effect of 5’4erminal phosphate upon the template activity of a doublet To determine whether correct recognition of two out of three bases in a codon may sometimes be sufficient to permit protein synthesis, the effect of the doublets pUpC and UpC upon the binding of [14C]Ser-sRNA to ribosomes was studied (Fig. 6(a)). The doublet with 5’4erminal phosphate, pUpC, stimulated Ser-sRNA binding to ribosomes, whereas UpC was without effect. The template activity of pUpC was proportional to the concentration within the range 0 to 60 mpmoles. To avoid contamination of pUpC with oligonucleotides of longer chain length, the doublet preparation was extensively purified before use (see Materials and Methods). No ultraviolet-absorbing contaminants were detected. The relationship between Mg2+ concentration and template activity of pUpC, UpC and UpCpU for Ser-sRNA is shown in Fig. 6(b). pUpC and UpCpU stimulated Ser-sRNA binding in reactions containing 0.02 to 0.08 M-Mg2+. The doublet UpC had no effect upon Ser-sRNA binding at each Mg2+ concentration tested. The specificity of pUpC and UpC for other [14C]-labeled AA-sRNA preparations which respond to triplets containing the sequence UpC was also studied. As shown in Table 3, pUpC stimulated the binding to ribosomes of Ser-sRNA but had no effect upon the binding of [14C]Leu-, and [14C]Ile-sRNA.
TEMPLATE
0
20
ACTIVITY
OF
40
80
60
Oligonucleotide
(mpmoles)
MODIFIED
0
RNA
CODONS
567
0.02 0.04 0.06 0.08 040 Concn McJ~+(M)
(a)
(b)
FIG. 6. The effect of UpC and pUpC on the binding of [‘%]Ser-sRNA to ribosomes. The relation between oligonucleotide concentration and [W]Ser-sRNA binding at 0.03 ar-Mg2 + is shown in (a). It should be noted that the ordinate begins at 1.25. The relation between (Mga+) concentration and [i%]Ser-sRNA binding is shown in (b). 50mpmoles of UPC, or pUpC, or 15 mpmolcs of UpCpU, were added to each reaction indicated. The remaining components of each 50-111.reaction included 0.420 Azao unit of sRNA acylated with 14.3 ppmoles of [i*C]serine plus the additions described in the legend of Fig. 1. Incubations were for 15 min at 24°C.
TABLET Effect of UpC and pUpC on AA-sRNA
SIX
Ile Val
63 mpmoles of either UpC or ponents of each 50-~1. reaction of the following AA-sRNA’s; [i4C]serine; [‘*C]Ile-sRNA, 0.34 sRNA, 0.32 A,,, unit acylated at 24°C.
None UPC PUPC None UPC PUPC None UPC PUPC
binding to ribosomes
1.45 1.53 1.98 0,22 0.31 0.25 0.34 0.29 0.31
pUpC were present in the reaction mixture. The remaining comincluded the additions described in the legend to Fig. 1 plus one [W]Ser-sRNA, 0.42 Azaa unit acylated with 14.3 pmnoles of A 2s0 unit acylated with 12.7 ppmoles of [‘%]isoleucine; [‘%]Valwith 16.8 ppmoles of [Wlvaline. Incubations were for 15 min
608
F.
ROTTMAN
AND
M.
NIRENBERG
These results show that a doublet with 5’-terminal phosphate, pUpC, directs Ser-sRNA binding to ribosomes. Ser-sRNA binds to ribosomes in response to triplets containing the sequence UpC in the first and second positions (UpCpU, UpCpC, UpCpA and UpCpG). UpC and pUpC did not stimulate binding of [14C]Val- or [14C]Ile-sRNA. These sRNA preparations respond to triplets containing the sequence UpC in the second and third positions. The data obtained in this study and related results obtained previously are summarized in Table 4. TABLET Relative template activity of substituted oligondeotides
OH (2’) OH (3’)
(5’)HO Template aotivityt relative to UpUpU
Oligonucleotide
p-5’-upupu UPUPU CHsO-pUpUpU upupu-3’-p UpUpUp-OCHs UpUpU-2’,3’-cyclic (2’-5’)-UpUpU Oligodeoxy T$
510 100 74 48 18 17 0 0
p
Template activity7 relative to ApApA
Oligonucleotide
p-5’-ApApA APAPA ApApA-3’-p ApApA-2*-p (2’-5’)-ApApA Oligodeoxy A$
181 100 51 15 0 0
t Relative template activities are approximations obtained by comparing the amount of AA-sRNA bound to ribosomes in the presence of limiting concentrations of oligonucleotides (0.50 mpmole of oligonucleotides containing U and 0.12 mpmole of oligonucleotides containing A) using the data presented in Figs 1, 2, 3 and 4. # Previous observations (Nirenberg & Leder, 1964).
4. Discussion Structural aspects of RNA codons which have been found to influence codon recognition include (3’~5’) phosphodiester linkages and free 2’-hydroxyl groups, since oligonucleotides with (2’~6’) phosphodiester linkages and oligonucleotides without 2’-hydroxyls (Nirenberg & Leder, 1964) have little or no template activity for AA-sRNA. Conversion of a S’-terminal phosphomonoester to a phosphodiester,
TEMPLATE
ACTIVITY
OF MODIFIED
RNA
CODONS
569
i.e. converting pUpUpU to either CHsO-pUpUpU or UpUpUpU, lowered template activity. Esterification of a terminal phosphomonoester with methanol enables one to examine the effects of adjacent phosphodiester linkages upon codon recognition without the introduction of nucleosides which might interact with ribosomes or sRNA. However, it is possible that the hydrophobic or steric character of the methyl group might influence template activity. The presence of a diester linkage on the 3’4erminus also inhibits template activity. This was observed with both UpUpUp-OCH, and UpUpU-2’,3’-cyclic phosphate. However, the latter compound represents both 2’- and 3’-terminal substitutions, and 2’- and 3’-terminal dies&s. The observed preference for 5’-terminal phosphomono- and diesters in comparison to 3’4erminal phosphomono- and dies&s may reflect a preferred polarity of interaction between the mRNA and the ribosome. Since 5’-terminal phosphate enhances template activity, it seems probable that a triphosphate attached to a 5’-terminal codon also would greatly affect template activity. RNA polymerase catalyzes the in vitro synthesis of mRNA with 5’terminal triphosphate (Maitra & Hurwitz, 1965; Bremer, Konrad, Gaines & Stem, 1965). The possibility that the 5’-terminus of mRNA specifies the attachment of the message to the ribosomes, selects the first word to be read, and thereby phases the reading, deserves to be emphasized. It should be noted that the terminal initiator codon may differ from an internal initiator codon in a polycistronic message, and similarly that a terminator codon at the end of an mRNA chain may differ from an internal terminator codon. It is possible that 5’- or 3’-terminal phosphomono- or diesters may impose restraints upon the orientation of terminal bases at ribosomal sites during codon recognition. Since one molecule of sRNA may recognize two or more synonym codons via alternate base pairing, it seems likely that terminal phosphate may differentially iniluence the template activity of synonym codons. The observation that 3’-terminal phosphate decreases the template activity of UpUpU but has little effect upon the activity of UpUpC is in accord with this suggestion. The effect of the doublet, pUpC, on Ser-sRNA binding demonstrates that certain for AA-sRNA. The effect of dinucleotides may have intrinsic template activity 5’-terminal phosphate deserves special emphasis, since UpC had no detectable template activity. Although it is clear that pUpC directs the binding of sRNA to ribosomes, we cannot distinguish between the recognition of one doublet or the overlapping recognition of three bases of adjacent doublets. It is particularly intriguing to relate the recognition of a doublet codon by sRNA to the evolution of the code. It is a pleasure to thank Miss Norma Zabriskie for her invaluable (F. R.) was supported by an American Cancer Society postdoctoral
assistance. One of us fellowship PF244.
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