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Involvement of specific ribosomal proteins in the aminoacyl-tRNA binding reaction
PRELIMINARY NOTES
555
BBA 91288
Involvement of specific ribosomal proteins in the ominoocyl-tRNA binding reoction Ribosomal proteins have been shown to function as binding sites for antibiotics and are believed to play participative roles in protein synthesis 1-a. Further, it has been reported 4 that free sulfhydryl groups of ribosomal proteins are required for poly U-directed binding of phenylalanyl-tRNA to mammalian ribosomes. As a first step toward clarifying the role of sulfhydryl groups in the binding reaction, we felt it would be rewarding to determine the uptake of radio-labeled iodoacetamide by ribosomes in various stages of complex formation. The rationale behind these experiments was to determine the iodoacetamide reactivity of specific electrophoretically separated protein fractions derived from free ribosomes and compare this with the uptake of iodoacetamide by those same proteins derived from ribosomes complexed with phenylalanyl-tRNA and/or poly U. Since at least one ribosomal sulfhydryl group is required for the binding of phenylalanyl-tRNA, it seemed likely that this thiol might be less accessible after formation of the ternary complex. This report presents evidence that the availability of certain iodoacetamide-reactive sites is indeed selectively influenced by the presence of prebound messenger and transfer RNA. In addition, there are several electrophoretically distinct protein fractions which do not seem to be involved in the binding process but which do appear to have iodoacetamide-reactive groups located on the exposed surface of the ribosome. Because of the large number of cysteine residues in ribosomal proteins, reproducible electrophoretic patterns could be obtained only after extensive alkylation of the proteins. The experimental procedure therefore demanded alkylation in two stages. The first reaction involved only those groups on the exposed surface of the ribosome; the second was more extensive, alkylating masked and buried thiols as well as those originally in disulfide linkage. Fig. I depicts the results of such an experiment in which iodo-I14C]acetamide was employed in the second reaction. Since any proteins masked by phenylalanyl-tRNA would not have reacted upon initial exposure to iodo-E12C]acetamide, we call this the direct method. Curve A shows a densitometric tracing of a typical stained gel, as well as a diagrammatic representation of the gel itself. As there were no observable differences in optical patterns from gel to gel, only one is presented in each figure. Such tracings were of course obtained for all gels and are omitted only for brevity and clarity of presentation. All bands can be located reproducibly by referring to the relative mobility scale which constitutes the abscissa for each graph. The protein apparently involved in binding is a dark band which exhibits a relative mobility of i . i and is designated A in the figures. Comparison of Fig. IB (free ribosomes) with IC (poly U-ribosome complex) and ID (phenylalanyl-tRNA-poly U-ribosome complex) clearly indicates that this band contains at least one protein which in the first step is more readily alkylated in the absence of phenylalanyl-tRNA. The inclusion of only poly U and ribosomes in the binding reaction does not significantly alter the reactivity of this protein fraction. The results of the converse experiment, in which the first rather than the second alkylation was done with iodo-[14C]acetamide, are shown in Fig. 2. As expected, the A band is labeled more extensively in the absence of phenylalanyl-tRNA. Biochim. Biophys. Acta, 2i 7 (197 o) 555-559
556
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Fig. I. U p t a k e of iodoE14C]acetamide b y ribosomal proteins: direct labeling method. KCl-extracted reticulocyte ribosomes (sRs) 5 were suspended in the buffered salts used for the poly U-directed " n o n e n z y m a t i c " binding of p h e n y l a l a n y l - t R N A 6. These ribosomes, either free, complexed with poly U or with b o t h poly U and p h e n y l a l a n y l - t R N A , were exposed to iodo~l~Clacetamide (final concent r a t i o n 2. 5 mM) for IO rain at 37 °, pH7. 4. Excess m e r c a p t o e t h a n o l was then added and the ribosomes were isolated b y centrifugation; t h e y were t h e n resuspended in 2 M LiC1- 4 M urea to precipitate ribosomal RNA. After centrifugation, the s u p e r n a t a n t was dialyzed at r o o m t e m p e r a t u r e against o.o2 M Tris-HC1, p H 8.o, in 8 M urea. The resulting solution was reacted with iodo[14Clacetamide (final concentration 2.5 mM) for 2o rain at 37 °. After adding excess mercaptoethanol, the m i x t u r e w a s dialyzed overnight in the cold against o.o3 M sodium acetate, p H 5.6, in 6 M urea. After polyacrylamide gel disc electrophoresis 7, densitometric tracings were made of the stained gels and mobility a s s i g n m e n t s made on the basis of a s h a r p and intense b a n d h a v i n g an actual mobility u n d e r these conditions of o.o6 cn12-V - 1 . h 1. The b a n d s were cut out, and their radioactivity d e t e r m i n e d b y liquid scintillation spectrometry. A. Densitometric tracing of a typical gel. B. Free (unconlplexed) ribosomes. C. Ribosomes plus poly U. D. Ribosomes plus poly U and phenylalanyl-tRNA.
We have previously reported ~ that sulfhydryl-reactive compounds fail to prevent binding of [3HJpoly U to reticulocyte ribosomes. Strangely, however, the complexing of poly U to ribosomes makes at least one site more available for alkylation Biochim. Biophys. Acta, 2i 7 (197 o) 555-559
PRELIMINARY NOTES
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Fig. 2. Uptake of iodo[14Cjacetamide by ribosomal proteins: indirect labeling method. The preparation and analytical procedures are identical to those described in Fig. i except that the order of exposure to radio-labeled iodoacetamide was reversed. That is, the ribosomes were first exposed to nonradioactive iodoacetamide and the ribosomal proteins isolated. The latter were further alkylated with iodo[14C~acetamide and then separated by electrophoresis and analyzed for radioactivity. A-D as in Fig. I. as shown b y e x a m i n a t i o n of the labeling p a t t e r n s in Figs. I a n d 2. Although the changes are less striking t h a n those exhibited b y the A b a n d , b o t h labeling m e t h o d s show t h a t the b a n d h a v i n g a relative m o b i l i t y of 0. 7 a n d designated M a c t u a l l y becomes more available for a l k y l a t i o n u p o n the a d d i t i o n of poly U. The "reclosure" of this site u p o n a d d i t i o n of t R N A (Figs I D a n d 2D) stimulates some i n t r i g u i n g speculations, none of which we can s u p p o r t at this time. The calculated changes in iodoacetamide r e a c t i v i t y due to the a d d i t i o n of p h e n y l a l a n y l - t R N A a n d / o r poly U can be f o u n d in Table I. These studies have revealed a n o t h e r p h e n o m e n o n of some interest, t h a t is the presence of several iodoacetamide-reactive groups on the surface of the ribosome Biochim. Biophys. Acta, 217 (197o) 555-559
558
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Biochim. Biophys. Acta, 2i 7 (i97 o) 555 559
o
PRELIMINARY NOTES
559
which do not seem to be involved in the binding reaction. That these proteins (designated $1-$4) contain groups readily available for alkylation can be seen by comparison of Figs. I and 2. Relative to other proteins within the same gel, these fractions are labeled more extensively by the indirect rather than the direct procedure. This indicates their relatively greater accessibility for reaction during the first exposure to iodoacetamide. In almost every case, the state of complexation of the ribosomes seems to have little effect. Inspection of Table I reveals an apparent inconsistency with regard to Fractions S~ and S 4 in that the observed absolute labeling is higher by the direct method. Our designation of these proteins as superficial is made on the basis t h a t over 2.5 times as much total akylation of ribosomal proteins occurs during the second exposure to iodoacetamide. We naturally appreciate that this does not preclude the possibility that only that portion of the protein containing the reactive group is on the surface, with the remainder of the polypeptide being buried in the interior of the ribosome. Our initial attempts to determine from which ribosomal subunit these various proteins are derived have been inconclusive because of co-electrophoresis of certain proteins from both subunits. We feel sufficiently confident, however, to tentatively assign Band $4 to the smaller and S 3 to the larger subunit. Further work is in progress to make definite assignments for all these bands. The skillful technical assistance of Mrs. A. S. Mostafapour is gratefully acknowledged. This research was supported in part by grants from the National Science Foundation (GB 7869) and the National Institutes of Health (AM 12710 ).
Department o/ Chemistry, Wayne State University, Detroit, Mich. 48202 ( U . S . A . ) I 2 3 4 5 6 7
TU-CHEN CHENG HARMON C. MCALLISTER
S. PESTKA, Proc. Natl. Acad. Sci. U.S., 64 (1969) 709. G. R. CRAVEN, R. GAVIN AND T. FANNING, Cold spring Harbor Syrup. Quant. Biol., 34 (I 969) 129 Y-B. CHAE, R. MAZUMDER AND S. OCHOA, Proc. Natl. Acad. Sci. U.S., 62 (1969) 1181. H. MCALLISTER AND R. SCHWEET,J. Mol. Biol., 34 (1968) 519. R. MILLER AND 1R. SCHWEET, Arch. Biochem. Biophys., 125 (1968) 632. R. HEINTZ, M. SALAS AND R. SCHWEET,Arch. Biochern. Biophys., t25 (1968) 488. R. REISFELD, U. LEWIS AND D. WILLIAMS, Nature, 195 (1962) 281.
Received July 6th, 197o Biochim. Biophys. Acta, 2i 7 (197 o) 555-559
555
BBA 91288
Involvement of specific ribosomal proteins in the ominoocyl-tRNA binding reoction Ribosomal proteins have been shown to function as binding sites for antibiotics and are believed to play participative roles in protein synthesis 1-a. Further, it has been reported 4 that free sulfhydryl groups of ribosomal proteins are required for poly U-directed binding of phenylalanyl-tRNA to mammalian ribosomes. As a first step toward clarifying the role of sulfhydryl groups in the binding reaction, we felt it would be rewarding to determine the uptake of radio-labeled iodoacetamide by ribosomes in various stages of complex formation. The rationale behind these experiments was to determine the iodoacetamide reactivity of specific electrophoretically separated protein fractions derived from free ribosomes and compare this with the uptake of iodoacetamide by those same proteins derived from ribosomes complexed with phenylalanyl-tRNA and/or poly U. Since at least one ribosomal sulfhydryl group is required for the binding of phenylalanyl-tRNA, it seemed likely that this thiol might be less accessible after formation of the ternary complex. This report presents evidence that the availability of certain iodoacetamide-reactive sites is indeed selectively influenced by the presence of prebound messenger and transfer RNA. In addition, there are several electrophoretically distinct protein fractions which do not seem to be involved in the binding process but which do appear to have iodoacetamide-reactive groups located on the exposed surface of the ribosome. Because of the large number of cysteine residues in ribosomal proteins, reproducible electrophoretic patterns could be obtained only after extensive alkylation of the proteins. The experimental procedure therefore demanded alkylation in two stages. The first reaction involved only those groups on the exposed surface of the ribosome; the second was more extensive, alkylating masked and buried thiols as well as those originally in disulfide linkage. Fig. I depicts the results of such an experiment in which iodo-I14C]acetamide was employed in the second reaction. Since any proteins masked by phenylalanyl-tRNA would not have reacted upon initial exposure to iodo-E12C]acetamide, we call this the direct method. Curve A shows a densitometric tracing of a typical stained gel, as well as a diagrammatic representation of the gel itself. As there were no observable differences in optical patterns from gel to gel, only one is presented in each figure. Such tracings were of course obtained for all gels and are omitted only for brevity and clarity of presentation. All bands can be located reproducibly by referring to the relative mobility scale which constitutes the abscissa for each graph. The protein apparently involved in binding is a dark band which exhibits a relative mobility of i . i and is designated A in the figures. Comparison of Fig. IB (free ribosomes) with IC (poly U-ribosome complex) and ID (phenylalanyl-tRNA-poly U-ribosome complex) clearly indicates that this band contains at least one protein which in the first step is more readily alkylated in the absence of phenylalanyl-tRNA. The inclusion of only poly U and ribosomes in the binding reaction does not significantly alter the reactivity of this protein fraction. The results of the converse experiment, in which the first rather than the second alkylation was done with iodo-[14C]acetamide, are shown in Fig. 2. As expected, the A band is labeled more extensively in the absence of phenylalanyl-tRNA. Biochim. Biophys. Acta, 2i 7 (197 o) 555-559
556
PRELIMINARY NOTES
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$2
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Fig. I. U p t a k e of iodoE14C]acetamide b y ribosomal proteins: direct labeling method. KCl-extracted reticulocyte ribosomes (sRs) 5 were suspended in the buffered salts used for the poly U-directed " n o n e n z y m a t i c " binding of p h e n y l a l a n y l - t R N A 6. These ribosomes, either free, complexed with poly U or with b o t h poly U and p h e n y l a l a n y l - t R N A , were exposed to iodo~l~Clacetamide (final concent r a t i o n 2. 5 mM) for IO rain at 37 °, pH7. 4. Excess m e r c a p t o e t h a n o l was then added and the ribosomes were isolated b y centrifugation; t h e y were t h e n resuspended in 2 M LiC1- 4 M urea to precipitate ribosomal RNA. After centrifugation, the s u p e r n a t a n t was dialyzed at r o o m t e m p e r a t u r e against o.o2 M Tris-HC1, p H 8.o, in 8 M urea. The resulting solution was reacted with iodo[14Clacetamide (final concentration 2.5 mM) for 2o rain at 37 °. After adding excess mercaptoethanol, the m i x t u r e w a s dialyzed overnight in the cold against o.o3 M sodium acetate, p H 5.6, in 6 M urea. After polyacrylamide gel disc electrophoresis 7, densitometric tracings were made of the stained gels and mobility a s s i g n m e n t s made on the basis of a s h a r p and intense b a n d h a v i n g an actual mobility u n d e r these conditions of o.o6 cn12-V - 1 . h 1. The b a n d s were cut out, and their radioactivity d e t e r m i n e d b y liquid scintillation spectrometry. A. Densitometric tracing of a typical gel. B. Free (unconlplexed) ribosomes. C. Ribosomes plus poly U. D. Ribosomes plus poly U and phenylalanyl-tRNA.
We have previously reported ~ that sulfhydryl-reactive compounds fail to prevent binding of [3HJpoly U to reticulocyte ribosomes. Strangely, however, the complexing of poly U to ribosomes makes at least one site more available for alkylation Biochim. Biophys. Acta, 2i 7 (197 o) 555-559
PRELIMINARY NOTES
A
557
B
160
M
A $7 E
120
C
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S,
$3
40
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, 0
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RELATIVEMOOILITY
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RELATIVEOiOOlLITY
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Fig. 2. Uptake of iodo[14Cjacetamide by ribosomal proteins: indirect labeling method. The preparation and analytical procedures are identical to those described in Fig. i except that the order of exposure to radio-labeled iodoacetamide was reversed. That is, the ribosomes were first exposed to nonradioactive iodoacetamide and the ribosomal proteins isolated. The latter were further alkylated with iodo[14C~acetamide and then separated by electrophoresis and analyzed for radioactivity. A-D as in Fig. I. as shown b y e x a m i n a t i o n of the labeling p a t t e r n s in Figs. I a n d 2. Although the changes are less striking t h a n those exhibited b y the A b a n d , b o t h labeling m e t h o d s show t h a t the b a n d h a v i n g a relative m o b i l i t y of 0. 7 a n d designated M a c t u a l l y becomes more available for a l k y l a t i o n u p o n the a d d i t i o n of poly U. The "reclosure" of this site u p o n a d d i t i o n of t R N A (Figs I D a n d 2D) stimulates some i n t r i g u i n g speculations, none of which we can s u p p o r t at this time. The calculated changes in iodoacetamide r e a c t i v i t y due to the a d d i t i o n of p h e n y l a l a n y l - t R N A a n d / o r poly U can be f o u n d in Table I. These studies have revealed a n o t h e r p h e n o m e n o n of some interest, t h a t is the presence of several iodoacetamide-reactive groups on the surface of the ribosome Biochim. Biophys. Acta, 217 (197o) 555-559
558
PRELIMINARY NOTES
i
.= ,n
I
e~g m
7
Z
~
~
"
2g o
÷
~
~.~ t
a ~ + ~
N
o o~ et
L
N
.,~+~ .~~
w
M
Biochim. Biophys. Acta, 2i 7 (i97 o) 555 559
o
PRELIMINARY NOTES
559
which do not seem to be involved in the binding reaction. That these proteins (designated $1-$4) contain groups readily available for alkylation can be seen by comparison of Figs. I and 2. Relative to other proteins within the same gel, these fractions are labeled more extensively by the indirect rather than the direct procedure. This indicates their relatively greater accessibility for reaction during the first exposure to iodoacetamide. In almost every case, the state of complexation of the ribosomes seems to have little effect. Inspection of Table I reveals an apparent inconsistency with regard to Fractions S~ and S 4 in that the observed absolute labeling is higher by the direct method. Our designation of these proteins as superficial is made on the basis t h a t over 2.5 times as much total akylation of ribosomal proteins occurs during the second exposure to iodoacetamide. We naturally appreciate that this does not preclude the possibility that only that portion of the protein containing the reactive group is on the surface, with the remainder of the polypeptide being buried in the interior of the ribosome. Our initial attempts to determine from which ribosomal subunit these various proteins are derived have been inconclusive because of co-electrophoresis of certain proteins from both subunits. We feel sufficiently confident, however, to tentatively assign Band $4 to the smaller and S 3 to the larger subunit. Further work is in progress to make definite assignments for all these bands. The skillful technical assistance of Mrs. A. S. Mostafapour is gratefully acknowledged. This research was supported in part by grants from the National Science Foundation (GB 7869) and the National Institutes of Health (AM 12710 ).
Department o/ Chemistry, Wayne State University, Detroit, Mich. 48202 ( U . S . A . ) I 2 3 4 5 6 7
TU-CHEN CHENG HARMON C. MCALLISTER
S. PESTKA, Proc. Natl. Acad. Sci. U.S., 64 (1969) 709. G. R. CRAVEN, R. GAVIN AND T. FANNING, Cold spring Harbor Syrup. Quant. Biol., 34 (I 969) 129 Y-B. CHAE, R. MAZUMDER AND S. OCHOA, Proc. Natl. Acad. Sci. U.S., 62 (1969) 1181. H. MCALLISTER AND R. SCHWEET,J. Mol. Biol., 34 (1968) 519. R. MILLER AND 1R. SCHWEET, Arch. Biochem. Biophys., 125 (1968) 632. R. HEINTZ, M. SALAS AND R. SCHWEET,Arch. Biochern. Biophys., t25 (1968) 488. R. REISFELD, U. LEWIS AND D. WILLIAMS, Nature, 195 (1962) 281.
Received July 6th, 197o Biochim. Biophys. Acta, 2i 7 (197 o) 555-559