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Interaction of ribosomes and some synthetic polyribonucleotides
PRELIMINARY NOTES
325
PN 6088 Interoction of ribosomes and some synthetic polyribonucleotides
NIRENBERG AND 1V[ATTHAEI1 first discovered in a cell-free system that the addition of poly-U specifically stimulated the incorporation of phenylalanine, and they suggested that the poly-U added functioned as a messenger RNA for polyphenylalanine synthesis. Thereafter, the function of a variety of synthetic polyribonucleotides was tested in their system in order to investigate the codes for other amino acids 2-5. It was found that they could accelerate the incorporation of other amino acids together with phenylalanine, provided that U was included in the copolymers. This synthetic polyribonucleotide-dependent amino acid-incorporation system appears to be most advantageous for analysing fundamental mechanism of protein synthesis on the ribosome. Since the first step of the over-'all react!on would be the interaction of ribosomes and the polyribonucleotides as the messenger RNA, we performed experiments to be described below. [s~PlPoly-U and [32P]poly-A were prepared enzyrnically from U-3~PP and A-a2PP with Micrococcus lysodeikticus polynucleotide phosphorylase (EC 2.7.7.8) purified b y the method of SINGER AND GUSS6. U-32PP and A-a2PP were synthesized according to SMITH A~D KHORANA?. The sedimentation coefficient of the poly-U synthesized was 8 S and that of po]y-A around 15 S. Ribosomes were prepared from Escherichia coli B(H) harvested in late log phase, and they were fractionated into 3o-S and 5o-S subunits by the method of TISSII~RES et al. s. The final preparations were dialysed against o.oi M Tris (pH 7.6) containing 6 mM mercaptoethanol and 5 mM Mg 2+ or 0.25 mM Mg~+, and kept in a refrigerator before use, without freezing. The enzyme fraction employed was the same as the Fraction $1oo of NIRENBERG AND MATTHAEI1. The poly-U-dependent incorporation of [l*Clphenylalanine into the ribosome preparations was tested, and it was demonstrated that only the recombine d ribosomes (I : 2 mixture of 3o-S and 5o-S subunits) stimulated the incorporation of phenylalanine, depending on poly-U added, whereas neither 3o-S or 5o-S subunit alone was capable of stimulating polyphenylalanine synthesis (Fig. i). Approx. i mg ribosomes and a varying quantity of [32P]poly-U were mixed in o.oi M Tris (pH 7.6) containing 0.25 mM or 5 m M Mg*+, and then the mixture was centrifuged in linear sucrose gradients (5-20 %, prepared in the same media), using Spinco rotor No. SW-25. The centrifugation was carried out at 25 ooo rev. per rain for 3 h. After the centrifugation, the distribution of the radioactivity, as the indicator of poly-U, and of the ultraviolet absorption in the centrifuge tubes was determined. In a medium containing 0.25 mM Mg~+, neither unfractionated ribosomes nor each separated subunit showed interaction with poly-U. At a Mg~+ concentration of 5 mM it was clearly demonstrated that either unfraetionated ribosomes or the 3o-S subunit interacted with poly-U yielding larger complexes. However, no interaction was observed with 5o-S subunit even in this Mg2+ concentration. In Fig. 2, (a) and (b) show the sedimentation patterns of each ribosome preparation and of synthesized Biochim. Biophys. Acta, 68 (1963) 325-327
326
PRELIMINARY NOTES
/
600
// /
f
:y /
//
L. 40C //
.c //
~ 2oc uo
/
/
20
40
Poly-U added (~g) Fig. I. Poly-U-dependent incorporation of E14C]phenylalanine into unfractionated ribosomes ( 0 - - - O ), ribosome fraction 30 S ( ~ - ~ ) , 5 ° S (q)-{)), and recombined ribosome fractions 3° S + 5o S (G-C)). Incubation system contained, in a final volume of 1 ml, about 0.6 mg of ribosomes, 1.2 mg of enzyme fraction, i / , m o l e of ATP, o . i / , m o l e each of GTP, CTP, 5/~moles of phosphoenolpyruvate, o.o 5 mg of pyruvate kinase (EC. 2.7.i.4o), o.o3/~mole of El~C]phenylalanine, o.o 5/~mole each of cold amino acid mixture (minus phenylalanine), I o / , m o l e s of magnesium acetate, 6o /~moles of KCI, 6/2moles of mercaptoethanol, IOO/*moles of Tris (pH 7.8) and a varying quantity of poly-U. Incubation lasted for 3 ° min at 37 °.
polyribonucleotides used in this study, and (c) to (e) those of the mixtures of each ribosome preparation and [8~P~poly-U, made in 5 mM Mg 2+. The formation of the ribosome-poly-U complex strikingly depended on the mixing ratio of ribosomes to poly-U(c/, the patterns of (e) to (g) in Fig. 2). If the quantity of poly-U added was in excess, the radioactivity distributed more closely to the 3o-S ribosome region. When the mixing ratio of poly-U to ribosomes was low, however, the bulk of the radioactivity distributed in the bottom layer, with a corresponding decrease of ultraviolet absorption of the 3o-S ribosome region. The same was observed with the mixture of unfractionated ribosomes and poly-U. This would mean that several ribosomes attached to a single molecule of poly-U, forming a cluster. It was again confirmed that no interaction occurred with the 5o-S subunit at any mixing ratio of ribosomes to poly-U. This observation suggests that the messenger RNA attached to ribosomes on the 3o-S subunit. As mentioned above, such interaction of poly-U and unfractionated ribosomes or 3o-S subunit depended on the Mg 2+ concentration, as in the case of the natural messenger RNA and ribosomesg, 1°. When a mixture of 3o-S subunit and poly-U prepared in 5 mM Mg 2+ was dialyzed against 0.25 mM Mg2+-o.oI M Tris (pH 7.6) a complete dissociation of the complex occurred (Fig. 2,h). Under similar experimental conditions, experiments were carried out with [3,pj_ poly-A in place of poly-U. Results of such experiments led to the striking conclusion that poly-A did not interact with any ribosomes even in 5 mM Mg*+. This was confirmed by repeated experiments. These observations appear to explain the previous finding 1-5 that poly-A did not stimulate the incorporation of any amino acid in the Nirenberg's system, apart from the question whether poly-A contains a code for an unknown amino acid or not. Studies are now being carried out on the detailed mechanism of the interaction between poly-U and ribosomes, and on the reason why poly-A Biochim. Biophys. Acta, 68 (1963) 325-327
PRELIMINARY NOTES
I
0 0,5"
ribosomes
[32p] poly-U
50S 30S
50 S + poty-UX1:0.15)
-~000
5raM Mg 2+
5 mM Mg 2+
4",
<6
I
%
'1
0,2.< 0.1 -
1o
I;
" 5T : "-,~ 10
2b
5ram Mg 2÷"
~
E
2"0
'15
Erection number
5ram
g
h
30 S + poly-U* (1:(~1)
30S+poly-U *t" 5ram Mg 2+ JP~
5raM Mg 2+
:
r
;
/
i
,]
,J
*
,
-4oo .C E
-200 c 0
30 S+ poly-U'~(1:03)
30S÷poly-U*(l:0.5)
0.2-
v
// f
~o.~-
o
,l
L
i
e 0,5-
-600 _~
/
'f
i
-BOO
I
!
u>' 0.3-
~
Unfractionated ribosomes +poly-U'{1:0.1)
5ram Mg2÷
5mM Mg2+~
0.4.
C
b
Unfroctionated
327
lO00
~0,25 mM Mg2+ll
800 "~
/I !i ooI
i
~ 0.1
ot
,
,
,
,
0
5
10
1b
20
I
,
,
,
,
•
10
15
20
5
10
15
20
; --;o
I'~
2'o
Froction number
Fig. 2. Interaction of ribosomes and E3*Y]poly-U. (a) and (b): Sedimentation patterns of individual ribosome preparations and synthesized polyribonucleotides, used in this study. (c) to (h): Sedimentation patterns of the mixtures of [8~P]poly-U and unfractionated ribosomes or their subunits. The values in the parentheses show the mixing ratio (w/w).
does n o t interact w i t h ribosomes, whereas p o l y - U does. It was r e c e n t l y f o u n d that poly-C, like p o l y - A , did n o t interact w i t h ribosomes. T h e a u t h o r s w i s h to t h a n k Professor A. SIBATANI for his helpful discussions.
Research Institute /or Nuclear Medicine and Biology, Hiroshima University, Hiroshima (Japan)
TOSHIO OKAMOTO
MITURU TAKANAMI
1 ~{. W. NIRENBERG AND J. H. MATTHAEI, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1588. G. lZ. MARTIN, J. H. MATTHAEI, O. W. JONES AND M. W. NIRENBERG, Biochem. Biophys. Res.
Commun., 6 (1962) 41o. 8 j . H. MATTHAEI, O. W. JONES, R. G. MARTIN AND M. \¥. ~N'IRENBERG, Proc. Natl. Acad. Sci. U.S., 48 (1962) 666. 4 p. LENGYEL, J. F. SPEYER AND S. OCHOA, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1936. s j . F. SPEYER, P. LENGYEL, C. BASlLIO AND S. OCHOA,Proc. Natl. Acad. Sci. U.S., 48 (1962) 63, 441. M. V. SINGER AND J. I~. G u s s , J. Biol. Chem., 237 (1962) 182. ? M. SMITH AND H. G. KHORANA, J. Am. Chem. Soc., 80 (1958) 1141. e A. TISSII~RES, J. I). WATSON, D. SCHLESSINGER AND B. 1~. HOLLINGWORTH, J. Mol. Biol., I (1959) 221. 9 M. NOMURA, B. D. HALL AND S. SPIEGELMAN, J . Mol. Biol., 2 (196o) 306. z0 A. ISHIHAMA, N. MIZUNO, N. TAKAI, E. OTAKA AND S. OSAWA, J. Mol. Biol., 5 (1962) 25I'
R e c e i v e d D e c e m b e r 29th, 1962 Biochim. Biophys. Acta, 68 (1963) 325-327
325
PN 6088 Interoction of ribosomes and some synthetic polyribonucleotides
NIRENBERG AND 1V[ATTHAEI1 first discovered in a cell-free system that the addition of poly-U specifically stimulated the incorporation of phenylalanine, and they suggested that the poly-U added functioned as a messenger RNA for polyphenylalanine synthesis. Thereafter, the function of a variety of synthetic polyribonucleotides was tested in their system in order to investigate the codes for other amino acids 2-5. It was found that they could accelerate the incorporation of other amino acids together with phenylalanine, provided that U was included in the copolymers. This synthetic polyribonucleotide-dependent amino acid-incorporation system appears to be most advantageous for analysing fundamental mechanism of protein synthesis on the ribosome. Since the first step of the over-'all react!on would be the interaction of ribosomes and the polyribonucleotides as the messenger RNA, we performed experiments to be described below. [s~PlPoly-U and [32P]poly-A were prepared enzyrnically from U-3~PP and A-a2PP with Micrococcus lysodeikticus polynucleotide phosphorylase (EC 2.7.7.8) purified b y the method of SINGER AND GUSS6. U-32PP and A-a2PP were synthesized according to SMITH A~D KHORANA?. The sedimentation coefficient of the poly-U synthesized was 8 S and that of po]y-A around 15 S. Ribosomes were prepared from Escherichia coli B(H) harvested in late log phase, and they were fractionated into 3o-S and 5o-S subunits by the method of TISSII~RES et al. s. The final preparations were dialysed against o.oi M Tris (pH 7.6) containing 6 mM mercaptoethanol and 5 mM Mg 2+ or 0.25 mM Mg~+, and kept in a refrigerator before use, without freezing. The enzyme fraction employed was the same as the Fraction $1oo of NIRENBERG AND MATTHAEI1. The poly-U-dependent incorporation of [l*Clphenylalanine into the ribosome preparations was tested, and it was demonstrated that only the recombine d ribosomes (I : 2 mixture of 3o-S and 5o-S subunits) stimulated the incorporation of phenylalanine, depending on poly-U added, whereas neither 3o-S or 5o-S subunit alone was capable of stimulating polyphenylalanine synthesis (Fig. i). Approx. i mg ribosomes and a varying quantity of [32P]poly-U were mixed in o.oi M Tris (pH 7.6) containing 0.25 mM or 5 m M Mg*+, and then the mixture was centrifuged in linear sucrose gradients (5-20 %, prepared in the same media), using Spinco rotor No. SW-25. The centrifugation was carried out at 25 ooo rev. per rain for 3 h. After the centrifugation, the distribution of the radioactivity, as the indicator of poly-U, and of the ultraviolet absorption in the centrifuge tubes was determined. In a medium containing 0.25 mM Mg~+, neither unfractionated ribosomes nor each separated subunit showed interaction with poly-U. At a Mg~+ concentration of 5 mM it was clearly demonstrated that either unfraetionated ribosomes or the 3o-S subunit interacted with poly-U yielding larger complexes. However, no interaction was observed with 5o-S subunit even in this Mg2+ concentration. In Fig. 2, (a) and (b) show the sedimentation patterns of each ribosome preparation and of synthesized Biochim. Biophys. Acta, 68 (1963) 325-327
326
PRELIMINARY NOTES
/
600
// /
f
:y /
//
L. 40C //
.c //
~ 2oc uo
/
/
20
40
Poly-U added (~g) Fig. I. Poly-U-dependent incorporation of E14C]phenylalanine into unfractionated ribosomes ( 0 - - - O ), ribosome fraction 30 S ( ~ - ~ ) , 5 ° S (q)-{)), and recombined ribosome fractions 3° S + 5o S (G-C)). Incubation system contained, in a final volume of 1 ml, about 0.6 mg of ribosomes, 1.2 mg of enzyme fraction, i / , m o l e of ATP, o . i / , m o l e each of GTP, CTP, 5/~moles of phosphoenolpyruvate, o.o 5 mg of pyruvate kinase (EC. 2.7.i.4o), o.o3/~mole of El~C]phenylalanine, o.o 5/~mole each of cold amino acid mixture (minus phenylalanine), I o / , m o l e s of magnesium acetate, 6o /~moles of KCI, 6/2moles of mercaptoethanol, IOO/*moles of Tris (pH 7.8) and a varying quantity of poly-U. Incubation lasted for 3 ° min at 37 °.
polyribonucleotides used in this study, and (c) to (e) those of the mixtures of each ribosome preparation and [8~P~poly-U, made in 5 mM Mg 2+. The formation of the ribosome-poly-U complex strikingly depended on the mixing ratio of ribosomes to poly-U(c/, the patterns of (e) to (g) in Fig. 2). If the quantity of poly-U added was in excess, the radioactivity distributed more closely to the 3o-S ribosome region. When the mixing ratio of poly-U to ribosomes was low, however, the bulk of the radioactivity distributed in the bottom layer, with a corresponding decrease of ultraviolet absorption of the 3o-S ribosome region. The same was observed with the mixture of unfractionated ribosomes and poly-U. This would mean that several ribosomes attached to a single molecule of poly-U, forming a cluster. It was again confirmed that no interaction occurred with the 5o-S subunit at any mixing ratio of ribosomes to poly-U. This observation suggests that the messenger RNA attached to ribosomes on the 3o-S subunit. As mentioned above, such interaction of poly-U and unfractionated ribosomes or 3o-S subunit depended on the Mg 2+ concentration, as in the case of the natural messenger RNA and ribosomesg, 1°. When a mixture of 3o-S subunit and poly-U prepared in 5 mM Mg 2+ was dialyzed against 0.25 mM Mg2+-o.oI M Tris (pH 7.6) a complete dissociation of the complex occurred (Fig. 2,h). Under similar experimental conditions, experiments were carried out with [3,pj_ poly-A in place of poly-U. Results of such experiments led to the striking conclusion that poly-A did not interact with any ribosomes even in 5 mM Mg*+. This was confirmed by repeated experiments. These observations appear to explain the previous finding 1-5 that poly-A did not stimulate the incorporation of any amino acid in the Nirenberg's system, apart from the question whether poly-A contains a code for an unknown amino acid or not. Studies are now being carried out on the detailed mechanism of the interaction between poly-U and ribosomes, and on the reason why poly-A Biochim. Biophys. Acta, 68 (1963) 325-327
PRELIMINARY NOTES
I
0 0,5"
ribosomes
[32p] poly-U
50S 30S
50 S + poty-UX1:0.15)
-~000
5raM Mg 2+
5 mM Mg 2+
4",
<6
I
%
'1
0,2.< 0.1 -
1o
I;
" 5T : "-,~ 10
2b
5ram Mg 2÷"
~
E
2"0
'15
Erection number
5ram
g
h
30 S + poly-U* (1:(~1)
30S+poly-U *t" 5ram Mg 2+ JP~
5raM Mg 2+
:
r
;
/
i
,]
,J
*
,
-4oo .C E
-200 c 0
30 S+ poly-U'~(1:03)
30S÷poly-U*(l:0.5)
0.2-
v
// f
~o.~-
o
,l
L
i
e 0,5-
-600 _~
/
'f
i
-BOO
I
!
u>' 0.3-
~
Unfractionated ribosomes +poly-U'{1:0.1)
5ram Mg2÷
5mM Mg2+~
0.4.
C
b
Unfroctionated
327
lO00
~0,25 mM Mg2+ll
800 "~
/I !i ooI
i
~ 0.1
ot
,
,
,
,
0
5
10
1b
20
I
,
,
,
,
•
10
15
20
5
10
15
20
; --;o
I'~
2'o
Froction number
Fig. 2. Interaction of ribosomes and E3*Y]poly-U. (a) and (b): Sedimentation patterns of individual ribosome preparations and synthesized polyribonucleotides, used in this study. (c) to (h): Sedimentation patterns of the mixtures of [8~P]poly-U and unfractionated ribosomes or their subunits. The values in the parentheses show the mixing ratio (w/w).
does n o t interact w i t h ribosomes, whereas p o l y - U does. It was r e c e n t l y f o u n d that poly-C, like p o l y - A , did n o t interact w i t h ribosomes. T h e a u t h o r s w i s h to t h a n k Professor A. SIBATANI for his helpful discussions.
Research Institute /or Nuclear Medicine and Biology, Hiroshima University, Hiroshima (Japan)
TOSHIO OKAMOTO
MITURU TAKANAMI
1 ~{. W. NIRENBERG AND J. H. MATTHAEI, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1588. G. lZ. MARTIN, J. H. MATTHAEI, O. W. JONES AND M. W. NIRENBERG, Biochem. Biophys. Res.
Commun., 6 (1962) 41o. 8 j . H. MATTHAEI, O. W. JONES, R. G. MARTIN AND M. \¥. ~N'IRENBERG, Proc. Natl. Acad. Sci. U.S., 48 (1962) 666. 4 p. LENGYEL, J. F. SPEYER AND S. OCHOA, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1936. s j . F. SPEYER, P. LENGYEL, C. BASlLIO AND S. OCHOA,Proc. Natl. Acad. Sci. U.S., 48 (1962) 63, 441. M. V. SINGER AND J. I~. G u s s , J. Biol. Chem., 237 (1962) 182. ? M. SMITH AND H. G. KHORANA, J. Am. Chem. Soc., 80 (1958) 1141. e A. TISSII~RES, J. I). WATSON, D. SCHLESSINGER AND B. 1~. HOLLINGWORTH, J. Mol. Biol., I (1959) 221. 9 M. NOMURA, B. D. HALL AND S. SPIEGELMAN, J . Mol. Biol., 2 (196o) 306. z0 A. ISHIHAMA, N. MIZUNO, N. TAKAI, E. OTAKA AND S. OSAWA, J. Mol. Biol., 5 (1962) 25I'
R e c e i v e d D e c e m b e r 29th, 1962 Biochim. Biophys. Acta, 68 (1963) 325-327