578
I N I T I A T I O N OF PROTEIN S Y N T H E S I S
[53] N u c l e o t i d e
Regulation
[53]
of Protein Synthesis
By GORDON M. WALTON and GORDON N. GILL l
Principle The utilization of GTP and subsequent hydrolysis to GDP and Pi during protein synthesis provides the primary source of energy for the initiation and elongation processes. Intracellular GTP levels for protein synthesis appear limiting except for its regeneration from the larger adenylate pool by reactions catalyzed by nucleoside diphosphate kinase and adenylate kinase (Fig. 1). Nucleoside diphosphate kinase catalyzes the phosphate transfer between nucleoside triphosphate and nucleoside diphosphate. Because the adenylate pool exceeds the guanylate pool, interphosphate transfer should have minor effects on the relative adenylate concentrations, and effects are primarily on guanylate concentrations. Adenylate kinase catalyzes the intraphosphate transfer between adenine nucleotides and catalyzes GTP: AMP phosphate transfer as well. Final concentrations of nucleotides depend on their relative concentrations and enzymic equilibrium constants. Eukaryotic initiation factor 2 (eIF-2) and eukaryotic elongation factor 1 (eEF- 1) have analogous functions in the initiation and elongation processes of polypeptide synthesis; both factors bind guanosine nucleotides and aminoacyl-tRNA, z-4 GDP.protein complexes are inactive in ternary and ribosomal complex formation. Thus, GTP.protein complexes are precursors of ternary and ribosomal complexes, and the GTP:GDP ratio determines the amount of ternary and subsequent ribosomal complex formation? In vitro studies of regulation of initiation and elongation complexes, directly by the GTP:GDP ratio, and indirectly by the adenylate pool (adenylate energy charge) help to elucidate the mechanisms controlling the rate of protein synthesis as it pertains to the availability of energy. 6 Procedures are provided for preparing materials and performing assays for the direct binding of nucleotides to eIF-2 and eIF-1 and the formation of ' Studies from the authors" laboratory were supported by U.S. Public Health Service Research Grant AM13149. 2 j. Lucas and F. Lipmann, Annu. Rev. Biochem. 40, 409 (1971). 3 j. M. Ravel, R. C. Dawkins, Jr., S. Lax, O. W. Odom, and B. Hardesty, Arch. Biochem. Biophys. 155, 332 (1973). 4 G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 390, 231 (1975). 5 G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 418, 195 (1976). 6 G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 447, 11 (1976).
METHODS IN ENZYMOLOGY, VOL. LX
Copyright © 1979by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4
[53]
NUCLEOTIDEREGULATIONOF PROTEIN SYNTHESIS
579
ENERGYPOOL ATP
ADP
AMP
GDP GTP
elF-2~
eEF-1
[elF-Z"GDP]Nucleotide[eEF-1.GDP]
[eIF-ZGTP]Complexes[eEF-I.GTP] MeI-~RNAf-~
~-- Phe-tRNl
[elF-2.GTP.Met-tRNAf] Ternory [eEF-1.GTP.Phe-tRNA] .~ Complexes [/,"80 S ribosome 40 S ribosome f poLy(U)
[Met-IRNAf40Srib°;me]CRi ompeb lxes°s°m°l[Phe[ tRNA80Sb°sir°me] INITIATION
ELONGATION
FIG. 1. Interaction of nucleotide pools and protein synthesis initiation and elongation complexes. AK, adenylate kinase; NDK, nucleoside diphosphate kinase. initiation and elongation complexes in the presence ofguanylate and adenylate pools. Materials for Assays N u c / e o t i d e s . GTP, GDP, [3H]GTP (10 Ci/mmol), and [3H]GDP (10 Ci/mmol) are purchased from commercial sources. The purity of nucleotides can be quickly and conveniently monitored by thin-layer chromatography on polyethylenimine cellulose as described by Randerath and Randerath. 7 Because commercial supplies of GTP and [3H]GTP are contaminated with GDP (approximately 3%), further purification is required. Similarly, [3H]GDP when chromatographed demonstrates the presence of an impurity (approximately 10%). To assure homogeneity of nucleotides, purification is achieved on DEAE-Sephadex A-25 with triethylammonium bicarbonate according to the procedure of Moffatt. ~ ATP, ADP, AMP, poly(A,G,U), and poly(U) are used as purchased from commercial sources. A m i n o a c y l - t R N A . Radiolabeled Met-tRNAf is synthesized from stripped yeast tRNA with [3H]methionine (5-15 Ci/mmol) or [3%]_
7 K. Randerath and E. Randerath, this series, Vol. 12A, p. 232. 8j. G. Moffatt,Can. J. Chem. 42, 599 (1964).
580
INITIATION OF PROTEIN SYNTHESIS
[53]
methionine (100-200 Ci/mmol) (New England Nuclear), using the procedure of Yang and NovellP and with Met-tRNAr synthetase prepared from E. coli B as described by RajBhandary and Gosh. 1° [3H]Phe-tRNA is synthesized from yeast-tRNA and [3H]phenylalanine (6 Ci/mmol) (Schwarz/Mann) using a rabbit reticulocyte enzyme preparation, rich in Phe-tRNA synthetase activity.ll 80 S Ribosomes and 40 S Ribosomal Subunits. Rabbit reticulocyte ribosomes and 40 S subunits are prepared according to the procedure of Schreier and Staehelin.l" The postribosomal supernatant of the reticulocyte lysate is utilized for the isolation of eEF- 1, and ribosomes are subsequently used for extraction of initiation factors and for 80 S ribosomal elongation complex formation. The 80 S ribosomes obtained contain significant levels ofeukaryotic elongation factor 2 (eEF-2) activity and require further purification before use in the Phe-tRNA binding assays. A useful procedure is that described by Takanami 13for the purification of ribosomes by MgCL precipitation. Even after this purification, some phenylalanine polymerization occurs (10-20%). Complete elimination of eEF-2 activity requires treatment with N-ethylmaleimide as described by Hardesty et al. 14 Purification o f Ribosomal Factors
The 0.5 M KCI ribosomal extract contains a number of proteins directly and indirectly involved in polypeptide synthesis, eIF-2 and nucleoside diphosphate kinase are examples, and their isolation and purification (along with another factor required for optimal 40 S ribosomal initiation complex formation) are described. Although methods for extensive purification have been described, 15-17 the procedures outlined conveniently provide factors of sufficient purity. Reagents
Buffer A: 20 mM Tris.HCl, pH 7.5, 1 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, 10% (v/v) glycerol Buffer A/0.2 M KCI Buffer A/0.4 M KC1 Buffer A/0.6 M KCI W. Yang and G. D. Novelli, this series, Vol. 20, p. 44. 1o U. R a j B h a n d a r y and H. P. Gosh, J. Biol. Chem. 244, 1104 (1969). " J. D. Irvin and B. H a r d e s t y , Biochemistry 11, 1915 (1972). 12 M. H. Schreier and T. Staehelin, J. Mol. Biol. 73, 329 (1973). 13 M. T a k a n a m i , Biochim. Biophys. Acta 39, 318 (1969). 14 B. H a r d e s t y , W. M c K e e h a n , and W. Culp, this series, Vol. 20, p. 316. 1~ L. M. Cashion, G. L. D e t t m a n , and W. M. Stanley, Jr., this series, Vol. 30, p. 153. 16 B. Safer, W. F. A n d e r s o n , and W. C. Merrick, J. Biol. Chem. 250, 9067 (1975). J7 R. Benne, C. Wong, M. Luedi, and J. W. B. H e r s h e y , J. Biol. Chem. 251, 7675 (1976).
[53]
N U C L E O T I D E R E G U L A T I O N OF P R O T E I N S Y N T H E S I S
581
Procedure. All purification steps are performed at 4°. Two milliliters of 4 M KC1 are added slowly with stirring to 14 ml of ribosomes (approximately 200 A 2~ounits/ml). The solution is centrifuged for 4 hr at 120,000 g. The supernatant is adjusted to 70% saturation by the slow addition of solid (NH4).,SO4 (7.5 g). The solution is centrifuged for 10 rain at 12,000g and the precipitate is dissolved in 4 ml of buffer A/0.2 M KC1 and dialyzed against 2 liters of the same buffer. After dialysis the preparation is centrifuged for 3 rain at 5000g to remove a small amount of insoluble precipitate. The clear solution, containing about 40 mg of protein, is applied to a phosphocellulose column (1.0 X 8.0 cm), equilibrated with buffer A/0.2 M KCI and washed free of unadsorbed protein with 2-3 column volumes of the same buffer. Two-milliliter fractions are collected and monitored by absorbances at A 2s0nm.The protein-containing fractions are pooled, concentrated to 1-2 ml in dialysis tubing against solid polyethylene glycol ( 15,000-20,000 MW) and dialyzed against 2 liters of buffer A/0.2 M KCI. Two additional protein fractions are eluted from the column in the same manner by a discontinuous gradient of buffer A/0.4 M KCI and buffer A/0.6 M KCI. These three preparations are stable for months stored in small aliquots at --70 °. The 0.2 M KCI fraction contains nucleoside diphosphate kinase that is further purified by gel filtration chromatography. Theprotein (15 mg in 0.5 ml) is applied to a Sephadex G-150 column (1.5 × 54 cm) equilibrated with buffer A/0.2 M KCI and eluted with the same buffer at a flow rate of 6 ml/hr. Fractions (3 ml) are collected and aliquots are assayed for nucleoside diphosphate kinase activity. The peak of activity is eluted at 1.4 void volumes and fractions 12-21 are pooled and concentrated. Protein yield was about 3 mg with a specific activity of 2. l units/rag. A unit of activity represents 1 p.mol of dGTP produced per minute per milligram of protein, as determined with the NADPH-dependent coupled assay system described by Mourad and Parks. ~ Alternatively, nucleoside diphosphate kinase can be assayed by the ATP-dependent formation of ternary initiation complex using GDP in place of GTP. 4 Nucleoside diphosphate kinase can also be obtained commercially at 80 units/mg (Boehringer Mannheim Biochemicals). The 0.4 M KCI fraction contains an initiation factor that stimulates Met-tRNAf binding to 40 S ribosomal subunits in a 40 S initiation complex assay. Protein yield is 6 mg and was used without further purification. The 0.6 M KCl fraction contains eIF-2 with a specific activity of 650700 units per milligram with a yield of about 3 mg. A unit of activity represents 1 pmol of Met-tRNAr bound in a GTP-dependent ternary initiation complex assay. Because eIF-2 has a greater affinity for GDP than GTP, recoveries from crude ribosomal fractions are difficult to determine be~ N. Mourad and R. E. Parks, Jr., J. Biol. Chem. 241,271 (1966).
582
INITIATION OF PROTEIN SYNTHESIS
[53]
cause they are rich in GTPase activity. This can be largely overcome when ternary complex formation is assayed in the presence of ATP. GTP regeneration can then occur through transphosphorylation catalyzed by nucleoside diphosphate kinase. GTPase activity in the 0.6 M KC1 fraction is insignificant. Purification o f eEF-I
eEF-1, which is found in the postribosomal supernatant of the rabbit reticulocyte lysate can be purified by a modification of the procedure described by Hardesty et al. 14 and Moidave et al. 19 Reagents Buffer A: 20 mM Tris.HCl, pH 8.0, I mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, 10% (v/v) glycerol Buffer A/0.05 M KCI Buffer A/0.1 M KCI Buffer A/0.4 M KCI Buffer B: 1 mM potassium phosphate, pH 6.8, 1 mM dithiothreitol, 10% (v/v) glycerol Buffer C: 150 mM potassium phosphate, pH 7.5, 1 mM dithiothreitol, 10% (v/v) glycerol Buffer D: 250 mM potassium phosphate, pH 7.5, 1 mM dithiothreitol, 10% (v/v) glycerol Procedure. All steps are performed at 4°. The postribosomal supernatant (500 ml) containing 50 mg of protein per milliliter is diluted with 0.5 volume of 10 mM Tris.HC1, pH 7.5, and while stirring, the protein is precipitated by slowly adding 350 g of(NH4)2SO4 with the pH maintained at 6.8 with 1 N acetic acid. The preparation is centrifuged at 12,000 g for 10 min. The precipitate is dissolved in 200 ml of buffer A/50 mM KCi and dialyzed against 6 liters of the same buffer. The protein is applied to a phosphocellulose column (2.5 × 45 cm) equilibrated with buffer A/50 mM KC1. The column is washed with 2 liters of buffer A/0.1 M KCI until free of protein as monitored at Ae80nm-eEF-1 activity is eluted with buffer A/0.4 M KCI. Fractions (10 ml) are collected and the protein peak (140-270 ml) is pooled, concentrated in dialysis tubing against solid polyethylene glycol to 10 ml, and dialyzed against 2 liters of buffer A/O. 1 M KC1. The yield is 130 mg of protein. The protein from phosphocellulose chromatography is applied to a Hypatite C (Clarkson Chemical Co.) column (1.5 × 30 cm) equilibrated with buffer B and washed free of protein with the same buffer. The column 19K. Moldave, W. Galasinski,and P. Rao, this series, Vol. 20, p. 337.
[53]
NUCLEOTIDE REGULATION OF PROTEIN SYNTHESIS
583
is then washed with buffer C followed by elution of eEF- l with buffer D. The eEF-l-containing fractions are pooled, concentrated, and dialyzed against 2 liters of buffer A/0.1 M KC1. The yield is about 15 mg of protein free of eEF-2 activity. Protein from hydroxyapatite chromatography is layered onto 14 ml of 15 to 37% linear glycerol gradients (5 mg/gradient) in buffer A/0.1 M KCI and centrifuged at 35,000 rpm for 20 hr in an SW 40 rotor (Beckman Instruments, Inc.). Twenty fractions (0.7 ml) are collected from the bottom and assayed for eEF-1 activity. Active fractions (11-15) are pooled, concentrated, and dialyzed against 1 liter of buffer A/0.1 M KC1. Protein yield is about I mg with a specific activity of 4300 units/mg without detectable levels of GTPase activity. One unit of activity equals 1 pmol of [3H]PhetRNA bound per milligram of protein in an 80 S ribosomal elongation complex assay. Contamination of either the ribosomes or eEF-1 preparations with eEF-2 results in polyphenylalanine synthesis with an apparent increase in eEF- 1 specific activity. A convenient test for polymerization is performed by separation of products bound to ribosomes on benzoylated diethylaminoethylcellulose as described by Pestka. TM Assay Procedures
Guanosine Nucleotide Binding Assays Direct binding studies of GTP and GDP with eIF-2 and eEF-l are summarized in Table I. eIF-2 has a greater affinity for GDP, while eEF-1 has a greater affinity for GTP. Reagents Buffer TKMD: 200 mM Tris.HC1 pH 7.5, 600 mM KCI, 10 mM MgCI2, 10 mM dithiothreitol Buffer TKM: 20 mM Tris.HCl pH 7.5, 60 mM KCI, 1 mM MgClz Buffer TNMD: 250 mM Tris .HCI pH 7.5, 800 mM NH4C1, 50 mM MgClz, 25 mM dithiothreitol Buffer TNM: 50 mM Tris.HC1, pH 7.5, 160 mM NH4C1, 10 mM MgC12 [3H]GTP, 10 p~M and 50 p~M, lithium salt [3H]GDP, 5 ~zM and 50 ~M, lithium salt Procedure for Determining elF-2 .Nucleotide Complex Formation. A complete reaction mixture in a final volume of 50/A contains 5/A of buffer TKMD, 0.7-2.8 units of purified eIF-2, ['~H]GTP, or [3H]GDP, and H20 to volume. After incubation at 30° for 10 min, the reaction is quantitatively 20S. Pestka, this series, Vol. 20, p. 508.
584
I N I T I A T I O N OF P R O T E I N S Y N T H E S I S
[53]
TABLE I SUMMARY OF NUCLEOTIDE AFFINITIES FOR RABBIT RETICULOCYTE elF-2 AND eEF-1 Factor
KDcDP
KDcTP
elF-2 'z eEF-1 ~
3.0 × 10-8M 9.2 × 10 6M
2.5 × 10-nM 4.2 × 10 7M
G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 390, 231 (1975). b G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 447, 11 (1976).
transferred to a Millipore filter reservoir containing 3 ml of cold buffer TKM. The reaction is filtered through Millipore filters (2.5 cm, 0.45/zm) which are rinsed 3 times with 5 ml of cold buffer TKM, dried for 10 min under a heat lamp, and counted in 5 ml of toluene-PPO/POPOP using a liquid scintillation counter. Procedure for Determining eEF-I .Nucleotide Complex Formation. A complete reaction mixture in a final volume of 50 t~l contains 10 ttl of buffer TNMD; 30-60 units of purified eEF-1, [3H]GTP, or [3H]GDP, and H20 to volume. After incubation at 30° for 10 min, Millipore filter retention of nucleotide complex is determined as described above except that filters are rinsed with buffer'TNM. Radioactivity bound to the filters in the absence of protein is determined and subtracted. Reactions proceed as well at 0°, but higher temperatures (30° or 37°) are desirable to facilitate the exchange of any bound nucleotide that may be associated with the isolated factor. Omission of exogenous MgCIz from the reaction mixture did not alter the affinity ofelF-2 for GDP or GTP, although a low (--10 -4 M) requirement has been suggested by Cashion et al.~5 Optimal conditions for binding of nucleotides to eEF-1 requires MgCl2 (10 AM) and NH4CI instead of KCI: even so, the affinity of eEF-I for GDP is extremely low and saturation of all binding sites by the nucleotide is not feasible with the Millipore filter assay.
Establishment of the Adenylate Energy Charge Adenylate energy charge is a metabolic regulatory parameter originally described by Atkinson. 21 Energy in the adenylate pool is defined as half the average number of anhydride-bound phosphate groups per adenine moiety, and in terms of the concentrations of the individual nucleotide equals ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP]). The energy charge index reflects the ratio of [ATP[/([AMP] + [ATP]) when ADP is absent. In vivo the equilibrium between the three nucleotides is catalyzed by adenylate kinase. eJ D. E. Atkinson, Biochemistry 7, 4030 (1968).
[53]
NUCLEOTIDE REGULATION OF PROTEIN SYNTHESIS
585
Initiation and elongation processes in protein synthesis require GTP and the potential for GTP regeneration from GDP and from the adenylate energy charge exists because nucleoside diphosphate kinase and adenylate kinase are abundant in ribosomal extracts.5 Establishment of an adenylate energy charge is performed as the initial step in a multistep assay prior to the addition of components involved in guanosine nucleotide complex formation and subsequent formation of initiation and elongation complexes. Reagents Buffer TKMD AMP, 20 raM, pH 7, potassium salt ATP, 20 raM, pH 7, potassium salt Adenylate kinase, 1000 units/rag (Sigma Chemical Co.) Procedure. A reaction mixture in a total volume of 20/~1 contains 5 p.1 of TKMD buffer; 5/zl of 20 mM AMP for an energy charge of zero or 5/A of 20 mM ATP for charge of 1.0; and 0.3 unit of adenylate kinase. Solutions containing various ratios of AMP and ATP at a total concentration of 20 mM are prepared to give the desired charge, e.g., 5 /zl of a solution containing 10 mM AMP and 10 mM ATP has a charge of 0.5. Incubation is at 30° for 10 rain and provides a 2 mM adenylate pool when the assay volume is subsequently raised to 50/~1 for ternary complex formation.
Ternary [Met-tRNA f GTP "elF-2 ] Initiation Complex Assay To demonstrate the effects of adenylate energy charge on the extent of ternary initiation complex formation, components of the ternary complex and nucleoside diphosphate kinase are added to the preincubated adenylate energy charge reaction mixture. After a second incubation, the GTPdependent Met-tRNA~ binding to eIF-2 is assayed by Millipore filter retention as described above for guanine nucleotide binding. Reagents Buffer TKMD Buffer TKM GTP, 1 mM, sodium salt ATP, 10 raM, pH 7.0, potassium salt [:~'~S]Met-tRNAf, 0.15 p.M Nucleoside diphosphate kinase, 2.1 units/mg eIF-2,650-700 units/mg Procedure. To 20/~1 of the preincubated adenylate energy charge reaction mixture the following components are added in a final reaction volume of 50 ~1:0.03 unit of nucleoside diphosphate kinase; 5/zl of I mM GTP; 5/~1 of 0.15 mM ['%S]Met-tRNA6 and 0.7-2.8 units of purified elF-2. The reaction is incubated at 30° for 10 min, and radioactivity bound to a
586
[53]
INITIATION OF PROTEIN SYNTHESIS
Millipore filter is determined. To measure activity in the absence of an adenylate energy charge, the charge reaction mixture is omitted and nucleoside diphosphate kinase is replaced with 5/zl of TKMD. When impure GTP is used in the assay in the absence of an adenylate energy charge, optimal activity is obtained by including ATP (5 /~1 of 10 mM) and nucleoside diphosphate kinase. Experiments designed to monitor the effects of the various components of the adenylate energy charge on ternary initiation complex formation at "various GTP:GDP ratios are summarized in Fig. 2. 5 With purified elF-2, exogenous magnesium ion had no significant effect on ternary complex formation in contrast to crude preparations that contain significant levels of magnesium-dependent GTPase activity giving rise to inhibitory levels of GDP. Magnesium ion is required, however, for adenylate kinase, nucleoside diphosphate kinase, and subsequent 40 S initiation complex formation and is therefore included.
40 S Ribosomal Initiation Complex Assay The second step in the sequential initiation assay, following ternary complex formation, is eIF-2-dependent Met-tRNAf binding to 40 S ribosomal subunits. Because the amount of ternary complex formation determines the extent of Met-tRNAf bound to 40 S subunits, the GTP:GDP ratio as regulated by adenylate energy charge will determine the extent of I
I
I
I
I
A. -NUCLEOSIDED/PHOSPHATE KIN'$E
z
1.2
0 123
GDP/GTP
I
i
I
F
~ ÷NUCLEOSIDEO/pHOSPHATE KIN,4SE
I
~ C.
I
/
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i
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i
- ADENYLATEKINASE~
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--Q . . . . . .
I
.~ ,-C," ~ ~
.Z z LLI
.-J 0 8
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0
/
v
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~A~NYLA[[KINASE o 02
0.4
06
08
1,0
02
0.4
06
08
I0
0
0,2
0.4
0.6
0.8
1.0
ENERGY CHARGE
FIG. 2. Effect of adenylate energy charge on ternary initiation complex formation. Total guanylate and adenylate pools were 0.1 mM and 2.0 mM, respectively. (B) Symbols correspond to the GDP:GTP ratios indicated in (A); (C) purified GTP alone was used. (A) Without nucleotide diphosphate kinase; (B) with nucleoside diphosphate kinase; (C) without adenylate kinase and nucleoside diphosphate kinase (D), plus adenylate kinase (HI), and plus adenylate kinase and nucleoside diphosphate kinase (0). From G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 418, 195 (1976).
[53]
N U C L E O T I D E REGULATION OF PROTEIN SYNTHESIS
587
40 S initiation complex formation. 6 The binding of [35S]Met-tRNAf to 40 S ribosomal subunits is analyzed by zone velocity sedimentation on preformed sucrose density gradients. Reagents Buffer TKMD 40 S ribosomal subunits, 50-100 A2G0units/ml Poly(A,G,U), 10 mg/ml MgCI.,, 10 mM Ribosomal factor in 0.4 M KCI fraction from phosphocellulose chromatography Sucrose gradient solutions: 15% and 37% (w/v) sucrose in 20 mM Tris • HCI, pH 7.5, 60 mM KC1, 2 mM MgCI,,, and 1 mM dithiothreitol Procedure. To 50/zl of the combined energy charge and ternary complex reaction mixture, the following components are added to give a final volume of 100/xl: 5/zl of buffer TKMD; 0.3 A.,60 unit of 40 S ribosomal subunits; 20/zg of poly(A,G,U); l0/zl of 10 mM MgC12; 26/zg of the 0.4M KCI fraction from phosphocellulose chromatography of ribosomal factors. After a 10-min incubation at 30°, the entire reaction mixture is layered at 4° on a 4-ml 15 to 37% linear sucrose gradient. Gradients are centrifuged at 45,000 rpm and 4° for 3 hr in an SW 56 rotor (Beckman Instruments, Inc.). Seventeen fractions of 0.24 ml are collected through the bottom of the tube into scintillation vials and radioactivity is determined in 10 ml of Bray's solution. Total radioactivity in the ['%S]Met-tRNAf.40 S subunit initiation complex (fractions 5 through 10) reflects the extent of complex formation. Met-tRNAf binding to 40 S ribosomal subunits is totally dependent on eIF-2 and stimulated approximately 3-fold by the 0.4 M KCI phosphocellulose fraction. Although activity observed at zero charge was 30% of that obtained at a charge of 1.0, 25% of total complex formation was observed in the absence of GTP. Poly(A,G,U) is not required for [35S]Met-tRNAf binding to 40 S ribosomal subunits under these conditions but does exert a slight stimulatory effect. The effect of adenylate energy charge on the extent of 40 S ribosomal initiation complex formation is shown in Fig. 3.
80 S Ribosomal Elongation Complex Assay To compare the effects of the adenylate energy charge on initiation complex formation to effects on elongation complex formation, GTPdependent binding of [3H]Phe-tRNA to 80 S ribosomes in the presence of varying adenylate energy charges was studied. The [3H]Phe-tRNA bound to ribosomes is determined by retention of the complex on Millipore filters as described by Hardesty et al. 14 and modified as outlined below.
588
INITIATION OF PROTEIN SYNTHESIS
"•v
I
I
I
I
0.2
0.4
0.6
0.8
[53]
100 ~ {:1:1 I--
8o-
60~
40-
I:L
20~L
0
1.0
ADENYLATE ENERGY CHARGE
Fro. 3. Effect of adenylate energy charge on 40 S ribosomal initiation and 80 S ribosomal elongation complex formation. Initiation complex was quantitated by sucrose gradient analysis and elongation complex by Millipore filter retention; 100%binding equals 0.42 and 1.18 pmol of [35S]Met-tRNAf(O) and [3H]Phe-tRNA(©), respectively. Adenylate pool was 2 mM, and guanylate pool was 0.1 mM. From G. M. Walton and G. N. Gill,Biochim. Biophys. Acta 447, 11 (1976). Reagents
Buffer T K M D Buffer T K M [3H]Phe-tRNA, 0 . 1 5 / x M GTP, 1 raM, sodium salt Poly(U), 10 mg/ml 80 S ribosomes, 100 A260 units/ml eEF-1, 4300 units/mg Procedure. To 20 ~1 of the adenylate energy charge reaction mixture the following components are added in a final reaction volume of 50/~1:5/xl of buffer T K M D ; 5/zl of 0.15 p~M [3H]Phe-tRNA; 5/xl of 1 m M GTP; 5 t~l of poly(U) (50/~g); 0.3 Az60 unit of 80 S ribosomes; and 1-3 units of e E F - I . After incubation at 37 ° for 30 min, the radioactivity bound is determined by Millipore filter retention as outlined for guanosine nucleotide complex formation. An assay blank is determined by omitting e E F - I . Figure 3 demonstrates the effect of the adenylate energy charge on both 40 S ribosomal initiation complex and 80 S ribosomal elongation complex formation. Preferential regulation of the initiation process is clearly evident as expected from the binding affinities ofeIF-2 and eEF- 1 for G T P and GDP (Table I). Remarks Adenine nucleotides do not directly effect formation of initiation and elongation complexes but exert indirect control through regulation of the
[53]
NUCLEOTIDE REGULATION OF PROTEIN SYNTHESIS
589
concentrations of GTP and GDP (Fig. 2C). In the absence of an adenylate pool, the GTP:GDP ratio determines the extent of initiation and elongation complex formation (Fig. 4). The broken line in Fig. 4 indicates the extent of 80 S elongation complex formation with GTP alone and demonstrates the GTPase activity of the 80 S ribosome. Magnesium ion-dependent inhibition of ternary initiation complex formation in crude ribosomal extracts appears to be the result of magnesiumdependent GTPase activity, because inhibition is overcome by GTP regeneration from ATP, and no inhibition is observed with purified elF-2 when GDP-free GTP is used. Further, the affinity of elF-2 for GTP or GDP was not appreciably affected by the presence or the absence of exogenous magnesium. 4 When a crude ribosomal extract is used as a source of initiation factors, the response to the adenylate energy is the same as that observed in the reconstructed system with purified components, e.g., 50% inhibition of ternary initiation complex formation at a charge of 0.85. ~ Because of the high affinity ofelF-2 for GDP, relative to GTP, minimal GTP hydrolysis gives rise to inhibitory levels of GDP. Elongation complex formation is less responsive to energy charge owing to the greater affinity of eEF-I for GTP, although some control is apparent with 50% inhibition observed at a charge of 0.60. In the presence of high adenylate energy charge, adenylate kinase and nucleoside diphosphate kinase, GTP regeneration occurs at the expense of ATP, while GDP levels are minimal and ribosomal complex formation remains optimal. At extremely low charge levels, GTP regeneration cannot occur, and, besides GTPase activity, I
r~
I---
I
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I00
f/
~== 6o 4o
n~ 20 o
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0.2
0.4
0.6
0.8
1.0
GTP GDP+GTP
FiG. 4. Effect of GTP mole fraction on ternary initiation complex and 80 S ribosomal elongation complex formation. Assays were by the Millipore filter procedure; 100% binding equals 1.04 and 2.15 pmol of [:~sS]Met-tRNAf (O) and [3H]Phe-tRNA (©), respectively. [~H]Phe-tRNA bound with GTP alone at concentrations used in mole fractions (0---©). Guanylate pool was 0.1 mM. From G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 447, 11 (1976).
590
INITIATION OF PROTEIN SYNTHESIS
[53]
energy in the guanylate pool is depleted by the greater adenylate pool through AMP:GTP transphosphorylation catalyzed by adenylate kinase. In vivo levels of nucleotides suggest that normally energy charge levels in the cell are in the range of 0.8-0.9, '''23 and the adenylate pool exceeds the guanylate pool by 10- to 20-fold. '4''5 As shown in Fig. 3, initiation complex formation is most sensitive to changes in the adenylate energy charge in the range found within the cell. A sensitive regulation of protein synthesis thus results from small changes in energy availability and links the overall rate of protein synthesis to the metabolic state of the cell.
~z A. 2:3 T. z4 A. z5 C.
G. C h a p m a n , L. Fall, and D. E. Atkinson, J. Bacteriol. 108, 1072 (1971). R. Live and E. K a m i n s k a s , J. Biol. Chem. 250, 1786 (1975). S. Bagnara and L. R. Finch, Eur. J. Biochem. 41,421 (1974). Colby and G. Edlin, Biochemistry. 9, 917 (1970).
I N I T I A T I O N OF PROTEIN S Y N T H E S I S
[53] N u c l e o t i d e
Regulation
[53]
of Protein Synthesis
By GORDON M. WALTON and GORDON N. GILL l
Principle The utilization of GTP and subsequent hydrolysis to GDP and Pi during protein synthesis provides the primary source of energy for the initiation and elongation processes. Intracellular GTP levels for protein synthesis appear limiting except for its regeneration from the larger adenylate pool by reactions catalyzed by nucleoside diphosphate kinase and adenylate kinase (Fig. 1). Nucleoside diphosphate kinase catalyzes the phosphate transfer between nucleoside triphosphate and nucleoside diphosphate. Because the adenylate pool exceeds the guanylate pool, interphosphate transfer should have minor effects on the relative adenylate concentrations, and effects are primarily on guanylate concentrations. Adenylate kinase catalyzes the intraphosphate transfer between adenine nucleotides and catalyzes GTP: AMP phosphate transfer as well. Final concentrations of nucleotides depend on their relative concentrations and enzymic equilibrium constants. Eukaryotic initiation factor 2 (eIF-2) and eukaryotic elongation factor 1 (eEF- 1) have analogous functions in the initiation and elongation processes of polypeptide synthesis; both factors bind guanosine nucleotides and aminoacyl-tRNA, z-4 GDP.protein complexes are inactive in ternary and ribosomal complex formation. Thus, GTP.protein complexes are precursors of ternary and ribosomal complexes, and the GTP:GDP ratio determines the amount of ternary and subsequent ribosomal complex formation? In vitro studies of regulation of initiation and elongation complexes, directly by the GTP:GDP ratio, and indirectly by the adenylate pool (adenylate energy charge) help to elucidate the mechanisms controlling the rate of protein synthesis as it pertains to the availability of energy. 6 Procedures are provided for preparing materials and performing assays for the direct binding of nucleotides to eIF-2 and eIF-1 and the formation of ' Studies from the authors" laboratory were supported by U.S. Public Health Service Research Grant AM13149. 2 j. Lucas and F. Lipmann, Annu. Rev. Biochem. 40, 409 (1971). 3 j. M. Ravel, R. C. Dawkins, Jr., S. Lax, O. W. Odom, and B. Hardesty, Arch. Biochem. Biophys. 155, 332 (1973). 4 G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 390, 231 (1975). 5 G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 418, 195 (1976). 6 G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 447, 11 (1976).
METHODS IN ENZYMOLOGY, VOL. LX
Copyright © 1979by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4
[53]
NUCLEOTIDEREGULATIONOF PROTEIN SYNTHESIS
579
ENERGYPOOL ATP
ADP
AMP
GDP GTP
elF-2~
eEF-1
[elF-Z"GDP]Nucleotide[eEF-1.GDP]
[eIF-ZGTP]Complexes[eEF-I.GTP] MeI-~RNAf-~
~-- Phe-tRNl
[elF-2.GTP.Met-tRNAf] Ternory [eEF-1.GTP.Phe-tRNA] .~ Complexes [/,"80 S ribosome 40 S ribosome f poLy(U)
[Met-IRNAf40Srib°;me]CRi ompeb lxes°s°m°l[Phe[ tRNA80Sb°sir°me] INITIATION
ELONGATION
FIG. 1. Interaction of nucleotide pools and protein synthesis initiation and elongation complexes. AK, adenylate kinase; NDK, nucleoside diphosphate kinase. initiation and elongation complexes in the presence ofguanylate and adenylate pools. Materials for Assays N u c / e o t i d e s . GTP, GDP, [3H]GTP (10 Ci/mmol), and [3H]GDP (10 Ci/mmol) are purchased from commercial sources. The purity of nucleotides can be quickly and conveniently monitored by thin-layer chromatography on polyethylenimine cellulose as described by Randerath and Randerath. 7 Because commercial supplies of GTP and [3H]GTP are contaminated with GDP (approximately 3%), further purification is required. Similarly, [3H]GDP when chromatographed demonstrates the presence of an impurity (approximately 10%). To assure homogeneity of nucleotides, purification is achieved on DEAE-Sephadex A-25 with triethylammonium bicarbonate according to the procedure of Moffatt. ~ ATP, ADP, AMP, poly(A,G,U), and poly(U) are used as purchased from commercial sources. A m i n o a c y l - t R N A . Radiolabeled Met-tRNAf is synthesized from stripped yeast tRNA with [3H]methionine (5-15 Ci/mmol) or [3%]_
7 K. Randerath and E. Randerath, this series, Vol. 12A, p. 232. 8j. G. Moffatt,Can. J. Chem. 42, 599 (1964).
580
INITIATION OF PROTEIN SYNTHESIS
[53]
methionine (100-200 Ci/mmol) (New England Nuclear), using the procedure of Yang and NovellP and with Met-tRNAr synthetase prepared from E. coli B as described by RajBhandary and Gosh. 1° [3H]Phe-tRNA is synthesized from yeast-tRNA and [3H]phenylalanine (6 Ci/mmol) (Schwarz/Mann) using a rabbit reticulocyte enzyme preparation, rich in Phe-tRNA synthetase activity.ll 80 S Ribosomes and 40 S Ribosomal Subunits. Rabbit reticulocyte ribosomes and 40 S subunits are prepared according to the procedure of Schreier and Staehelin.l" The postribosomal supernatant of the reticulocyte lysate is utilized for the isolation of eEF- 1, and ribosomes are subsequently used for extraction of initiation factors and for 80 S ribosomal elongation complex formation. The 80 S ribosomes obtained contain significant levels ofeukaryotic elongation factor 2 (eEF-2) activity and require further purification before use in the Phe-tRNA binding assays. A useful procedure is that described by Takanami 13for the purification of ribosomes by MgCL precipitation. Even after this purification, some phenylalanine polymerization occurs (10-20%). Complete elimination of eEF-2 activity requires treatment with N-ethylmaleimide as described by Hardesty et al. 14 Purification o f Ribosomal Factors
The 0.5 M KCI ribosomal extract contains a number of proteins directly and indirectly involved in polypeptide synthesis, eIF-2 and nucleoside diphosphate kinase are examples, and their isolation and purification (along with another factor required for optimal 40 S ribosomal initiation complex formation) are described. Although methods for extensive purification have been described, 15-17 the procedures outlined conveniently provide factors of sufficient purity. Reagents
Buffer A: 20 mM Tris.HCl, pH 7.5, 1 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, 10% (v/v) glycerol Buffer A/0.2 M KCI Buffer A/0.4 M KC1 Buffer A/0.6 M KCI W. Yang and G. D. Novelli, this series, Vol. 20, p. 44. 1o U. R a j B h a n d a r y and H. P. Gosh, J. Biol. Chem. 244, 1104 (1969). " J. D. Irvin and B. H a r d e s t y , Biochemistry 11, 1915 (1972). 12 M. H. Schreier and T. Staehelin, J. Mol. Biol. 73, 329 (1973). 13 M. T a k a n a m i , Biochim. Biophys. Acta 39, 318 (1969). 14 B. H a r d e s t y , W. M c K e e h a n , and W. Culp, this series, Vol. 20, p. 316. 1~ L. M. Cashion, G. L. D e t t m a n , and W. M. Stanley, Jr., this series, Vol. 30, p. 153. 16 B. Safer, W. F. A n d e r s o n , and W. C. Merrick, J. Biol. Chem. 250, 9067 (1975). J7 R. Benne, C. Wong, M. Luedi, and J. W. B. H e r s h e y , J. Biol. Chem. 251, 7675 (1976).
[53]
N U C L E O T I D E R E G U L A T I O N OF P R O T E I N S Y N T H E S I S
581
Procedure. All purification steps are performed at 4°. Two milliliters of 4 M KC1 are added slowly with stirring to 14 ml of ribosomes (approximately 200 A 2~ounits/ml). The solution is centrifuged for 4 hr at 120,000 g. The supernatant is adjusted to 70% saturation by the slow addition of solid (NH4).,SO4 (7.5 g). The solution is centrifuged for 10 rain at 12,000g and the precipitate is dissolved in 4 ml of buffer A/0.2 M KC1 and dialyzed against 2 liters of the same buffer. After dialysis the preparation is centrifuged for 3 rain at 5000g to remove a small amount of insoluble precipitate. The clear solution, containing about 40 mg of protein, is applied to a phosphocellulose column (1.0 X 8.0 cm), equilibrated with buffer A/0.2 M KCI and washed free of unadsorbed protein with 2-3 column volumes of the same buffer. Two-milliliter fractions are collected and monitored by absorbances at A 2s0nm.The protein-containing fractions are pooled, concentrated to 1-2 ml in dialysis tubing against solid polyethylene glycol ( 15,000-20,000 MW) and dialyzed against 2 liters of buffer A/0.2 M KCI. Two additional protein fractions are eluted from the column in the same manner by a discontinuous gradient of buffer A/0.4 M KCI and buffer A/0.6 M KCI. These three preparations are stable for months stored in small aliquots at --70 °. The 0.2 M KCI fraction contains nucleoside diphosphate kinase that is further purified by gel filtration chromatography. Theprotein (15 mg in 0.5 ml) is applied to a Sephadex G-150 column (1.5 × 54 cm) equilibrated with buffer A/0.2 M KCI and eluted with the same buffer at a flow rate of 6 ml/hr. Fractions (3 ml) are collected and aliquots are assayed for nucleoside diphosphate kinase activity. The peak of activity is eluted at 1.4 void volumes and fractions 12-21 are pooled and concentrated. Protein yield was about 3 mg with a specific activity of 2. l units/rag. A unit of activity represents 1 p.mol of dGTP produced per minute per milligram of protein, as determined with the NADPH-dependent coupled assay system described by Mourad and Parks. ~ Alternatively, nucleoside diphosphate kinase can be assayed by the ATP-dependent formation of ternary initiation complex using GDP in place of GTP. 4 Nucleoside diphosphate kinase can also be obtained commercially at 80 units/mg (Boehringer Mannheim Biochemicals). The 0.4 M KCI fraction contains an initiation factor that stimulates Met-tRNAf binding to 40 S ribosomal subunits in a 40 S initiation complex assay. Protein yield is 6 mg and was used without further purification. The 0.6 M KCl fraction contains eIF-2 with a specific activity of 650700 units per milligram with a yield of about 3 mg. A unit of activity represents 1 pmol of Met-tRNAr bound in a GTP-dependent ternary initiation complex assay. Because eIF-2 has a greater affinity for GDP than GTP, recoveries from crude ribosomal fractions are difficult to determine be~ N. Mourad and R. E. Parks, Jr., J. Biol. Chem. 241,271 (1966).
582
INITIATION OF PROTEIN SYNTHESIS
[53]
cause they are rich in GTPase activity. This can be largely overcome when ternary complex formation is assayed in the presence of ATP. GTP regeneration can then occur through transphosphorylation catalyzed by nucleoside diphosphate kinase. GTPase activity in the 0.6 M KC1 fraction is insignificant. Purification o f eEF-I
eEF-1, which is found in the postribosomal supernatant of the rabbit reticulocyte lysate can be purified by a modification of the procedure described by Hardesty et al. 14 and Moidave et al. 19 Reagents Buffer A: 20 mM Tris.HCl, pH 8.0, I mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, 10% (v/v) glycerol Buffer A/0.05 M KCI Buffer A/0.1 M KCI Buffer A/0.4 M KCI Buffer B: 1 mM potassium phosphate, pH 6.8, 1 mM dithiothreitol, 10% (v/v) glycerol Buffer C: 150 mM potassium phosphate, pH 7.5, 1 mM dithiothreitol, 10% (v/v) glycerol Buffer D: 250 mM potassium phosphate, pH 7.5, 1 mM dithiothreitol, 10% (v/v) glycerol Procedure. All steps are performed at 4°. The postribosomal supernatant (500 ml) containing 50 mg of protein per milliliter is diluted with 0.5 volume of 10 mM Tris.HC1, pH 7.5, and while stirring, the protein is precipitated by slowly adding 350 g of(NH4)2SO4 with the pH maintained at 6.8 with 1 N acetic acid. The preparation is centrifuged at 12,000 g for 10 min. The precipitate is dissolved in 200 ml of buffer A/50 mM KCi and dialyzed against 6 liters of the same buffer. The protein is applied to a phosphocellulose column (2.5 × 45 cm) equilibrated with buffer A/50 mM KC1. The column is washed with 2 liters of buffer A/0.1 M KCI until free of protein as monitored at Ae80nm-eEF-1 activity is eluted with buffer A/0.4 M KCI. Fractions (10 ml) are collected and the protein peak (140-270 ml) is pooled, concentrated in dialysis tubing against solid polyethylene glycol to 10 ml, and dialyzed against 2 liters of buffer A/O. 1 M KC1. The yield is 130 mg of protein. The protein from phosphocellulose chromatography is applied to a Hypatite C (Clarkson Chemical Co.) column (1.5 × 30 cm) equilibrated with buffer B and washed free of protein with the same buffer. The column 19K. Moldave, W. Galasinski,and P. Rao, this series, Vol. 20, p. 337.
[53]
NUCLEOTIDE REGULATION OF PROTEIN SYNTHESIS
583
is then washed with buffer C followed by elution of eEF- l with buffer D. The eEF-l-containing fractions are pooled, concentrated, and dialyzed against 2 liters of buffer A/0.1 M KC1. The yield is about 15 mg of protein free of eEF-2 activity. Protein from hydroxyapatite chromatography is layered onto 14 ml of 15 to 37% linear glycerol gradients (5 mg/gradient) in buffer A/0.1 M KCI and centrifuged at 35,000 rpm for 20 hr in an SW 40 rotor (Beckman Instruments, Inc.). Twenty fractions (0.7 ml) are collected from the bottom and assayed for eEF-1 activity. Active fractions (11-15) are pooled, concentrated, and dialyzed against 1 liter of buffer A/0.1 M KC1. Protein yield is about I mg with a specific activity of 4300 units/mg without detectable levels of GTPase activity. One unit of activity equals 1 pmol of [3H]PhetRNA bound per milligram of protein in an 80 S ribosomal elongation complex assay. Contamination of either the ribosomes or eEF-1 preparations with eEF-2 results in polyphenylalanine synthesis with an apparent increase in eEF- 1 specific activity. A convenient test for polymerization is performed by separation of products bound to ribosomes on benzoylated diethylaminoethylcellulose as described by Pestka. TM Assay Procedures
Guanosine Nucleotide Binding Assays Direct binding studies of GTP and GDP with eIF-2 and eEF-l are summarized in Table I. eIF-2 has a greater affinity for GDP, while eEF-1 has a greater affinity for GTP. Reagents Buffer TKMD: 200 mM Tris.HC1 pH 7.5, 600 mM KCI, 10 mM MgCI2, 10 mM dithiothreitol Buffer TKM: 20 mM Tris.HCl pH 7.5, 60 mM KCI, 1 mM MgClz Buffer TNMD: 250 mM Tris .HCI pH 7.5, 800 mM NH4C1, 50 mM MgClz, 25 mM dithiothreitol Buffer TNM: 50 mM Tris.HC1, pH 7.5, 160 mM NH4C1, 10 mM MgC12 [3H]GTP, 10 p~M and 50 p~M, lithium salt [3H]GDP, 5 ~zM and 50 ~M, lithium salt Procedure for Determining elF-2 .Nucleotide Complex Formation. A complete reaction mixture in a final volume of 50/A contains 5/A of buffer TKMD, 0.7-2.8 units of purified eIF-2, ['~H]GTP, or [3H]GDP, and H20 to volume. After incubation at 30° for 10 min, the reaction is quantitatively 20S. Pestka, this series, Vol. 20, p. 508.
584
I N I T I A T I O N OF P R O T E I N S Y N T H E S I S
[53]
TABLE I SUMMARY OF NUCLEOTIDE AFFINITIES FOR RABBIT RETICULOCYTE elF-2 AND eEF-1 Factor
KDcDP
KDcTP
elF-2 'z eEF-1 ~
3.0 × 10-8M 9.2 × 10 6M
2.5 × 10-nM 4.2 × 10 7M
G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 390, 231 (1975). b G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 447, 11 (1976).
transferred to a Millipore filter reservoir containing 3 ml of cold buffer TKM. The reaction is filtered through Millipore filters (2.5 cm, 0.45/zm) which are rinsed 3 times with 5 ml of cold buffer TKM, dried for 10 min under a heat lamp, and counted in 5 ml of toluene-PPO/POPOP using a liquid scintillation counter. Procedure for Determining eEF-I .Nucleotide Complex Formation. A complete reaction mixture in a final volume of 50 t~l contains 10 ttl of buffer TNMD; 30-60 units of purified eEF-1, [3H]GTP, or [3H]GDP, and H20 to volume. After incubation at 30° for 10 min, Millipore filter retention of nucleotide complex is determined as described above except that filters are rinsed with buffer'TNM. Radioactivity bound to the filters in the absence of protein is determined and subtracted. Reactions proceed as well at 0°, but higher temperatures (30° or 37°) are desirable to facilitate the exchange of any bound nucleotide that may be associated with the isolated factor. Omission of exogenous MgCIz from the reaction mixture did not alter the affinity ofelF-2 for GDP or GTP, although a low (--10 -4 M) requirement has been suggested by Cashion et al.~5 Optimal conditions for binding of nucleotides to eEF-1 requires MgCl2 (10 AM) and NH4CI instead of KCI: even so, the affinity of eEF-I for GDP is extremely low and saturation of all binding sites by the nucleotide is not feasible with the Millipore filter assay.
Establishment of the Adenylate Energy Charge Adenylate energy charge is a metabolic regulatory parameter originally described by Atkinson. 21 Energy in the adenylate pool is defined as half the average number of anhydride-bound phosphate groups per adenine moiety, and in terms of the concentrations of the individual nucleotide equals ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP]). The energy charge index reflects the ratio of [ATP[/([AMP] + [ATP]) when ADP is absent. In vivo the equilibrium between the three nucleotides is catalyzed by adenylate kinase. eJ D. E. Atkinson, Biochemistry 7, 4030 (1968).
[53]
NUCLEOTIDE REGULATION OF PROTEIN SYNTHESIS
585
Initiation and elongation processes in protein synthesis require GTP and the potential for GTP regeneration from GDP and from the adenylate energy charge exists because nucleoside diphosphate kinase and adenylate kinase are abundant in ribosomal extracts.5 Establishment of an adenylate energy charge is performed as the initial step in a multistep assay prior to the addition of components involved in guanosine nucleotide complex formation and subsequent formation of initiation and elongation complexes. Reagents Buffer TKMD AMP, 20 raM, pH 7, potassium salt ATP, 20 raM, pH 7, potassium salt Adenylate kinase, 1000 units/rag (Sigma Chemical Co.) Procedure. A reaction mixture in a total volume of 20/~1 contains 5 p.1 of TKMD buffer; 5/zl of 20 mM AMP for an energy charge of zero or 5/A of 20 mM ATP for charge of 1.0; and 0.3 unit of adenylate kinase. Solutions containing various ratios of AMP and ATP at a total concentration of 20 mM are prepared to give the desired charge, e.g., 5 /zl of a solution containing 10 mM AMP and 10 mM ATP has a charge of 0.5. Incubation is at 30° for 10 rain and provides a 2 mM adenylate pool when the assay volume is subsequently raised to 50/~1 for ternary complex formation.
Ternary [Met-tRNA f GTP "elF-2 ] Initiation Complex Assay To demonstrate the effects of adenylate energy charge on the extent of ternary initiation complex formation, components of the ternary complex and nucleoside diphosphate kinase are added to the preincubated adenylate energy charge reaction mixture. After a second incubation, the GTPdependent Met-tRNA~ binding to eIF-2 is assayed by Millipore filter retention as described above for guanine nucleotide binding. Reagents Buffer TKMD Buffer TKM GTP, 1 mM, sodium salt ATP, 10 raM, pH 7.0, potassium salt [:~'~S]Met-tRNAf, 0.15 p.M Nucleoside diphosphate kinase, 2.1 units/mg eIF-2,650-700 units/mg Procedure. To 20/~1 of the preincubated adenylate energy charge reaction mixture the following components are added in a final reaction volume of 50 ~1:0.03 unit of nucleoside diphosphate kinase; 5/zl of I mM GTP; 5/~1 of 0.15 mM ['%S]Met-tRNA6 and 0.7-2.8 units of purified elF-2. The reaction is incubated at 30° for 10 min, and radioactivity bound to a
586
[53]
INITIATION OF PROTEIN SYNTHESIS
Millipore filter is determined. To measure activity in the absence of an adenylate energy charge, the charge reaction mixture is omitted and nucleoside diphosphate kinase is replaced with 5/zl of TKMD. When impure GTP is used in the assay in the absence of an adenylate energy charge, optimal activity is obtained by including ATP (5 /~1 of 10 mM) and nucleoside diphosphate kinase. Experiments designed to monitor the effects of the various components of the adenylate energy charge on ternary initiation complex formation at "various GTP:GDP ratios are summarized in Fig. 2. 5 With purified elF-2, exogenous magnesium ion had no significant effect on ternary complex formation in contrast to crude preparations that contain significant levels of magnesium-dependent GTPase activity giving rise to inhibitory levels of GDP. Magnesium ion is required, however, for adenylate kinase, nucleoside diphosphate kinase, and subsequent 40 S initiation complex formation and is therefore included.
40 S Ribosomal Initiation Complex Assay The second step in the sequential initiation assay, following ternary complex formation, is eIF-2-dependent Met-tRNAf binding to 40 S ribosomal subunits. Because the amount of ternary complex formation determines the extent of Met-tRNAf bound to 40 S subunits, the GTP:GDP ratio as regulated by adenylate energy charge will determine the extent of I
I
I
I
I
A. -NUCLEOSIDED/PHOSPHATE KIN'$E
z
1.2
0 123
GDP/GTP
I
i
I
F
~ ÷NUCLEOSIDEO/pHOSPHATE KIN,4SE
I
~ C.
I
/
I
i
I
i
- ADENYLATEKINASE~
D" . . . . .
--Q . . . . . .
I
.~ ,-C," ~ ~
.Z z LLI
.-J 0 8
I0 -2
0
/
v
0.4 -r" r~
~A~NYLA[[KINASE o 02
0.4
06
08
1,0
02
0.4
06
08
I0
0
0,2
0.4
0.6
0.8
1.0
ENERGY CHARGE
FIG. 2. Effect of adenylate energy charge on ternary initiation complex formation. Total guanylate and adenylate pools were 0.1 mM and 2.0 mM, respectively. (B) Symbols correspond to the GDP:GTP ratios indicated in (A); (C) purified GTP alone was used. (A) Without nucleotide diphosphate kinase; (B) with nucleoside diphosphate kinase; (C) without adenylate kinase and nucleoside diphosphate kinase (D), plus adenylate kinase (HI), and plus adenylate kinase and nucleoside diphosphate kinase (0). From G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 418, 195 (1976).
[53]
N U C L E O T I D E REGULATION OF PROTEIN SYNTHESIS
587
40 S initiation complex formation. 6 The binding of [35S]Met-tRNAf to 40 S ribosomal subunits is analyzed by zone velocity sedimentation on preformed sucrose density gradients. Reagents Buffer TKMD 40 S ribosomal subunits, 50-100 A2G0units/ml Poly(A,G,U), 10 mg/ml MgCI.,, 10 mM Ribosomal factor in 0.4 M KCI fraction from phosphocellulose chromatography Sucrose gradient solutions: 15% and 37% (w/v) sucrose in 20 mM Tris • HCI, pH 7.5, 60 mM KC1, 2 mM MgCI,,, and 1 mM dithiothreitol Procedure. To 50/zl of the combined energy charge and ternary complex reaction mixture, the following components are added to give a final volume of 100/xl: 5/zl of buffer TKMD; 0.3 A.,60 unit of 40 S ribosomal subunits; 20/zg of poly(A,G,U); l0/zl of 10 mM MgC12; 26/zg of the 0.4M KCI fraction from phosphocellulose chromatography of ribosomal factors. After a 10-min incubation at 30°, the entire reaction mixture is layered at 4° on a 4-ml 15 to 37% linear sucrose gradient. Gradients are centrifuged at 45,000 rpm and 4° for 3 hr in an SW 56 rotor (Beckman Instruments, Inc.). Seventeen fractions of 0.24 ml are collected through the bottom of the tube into scintillation vials and radioactivity is determined in 10 ml of Bray's solution. Total radioactivity in the ['%S]Met-tRNAf.40 S subunit initiation complex (fractions 5 through 10) reflects the extent of complex formation. Met-tRNAf binding to 40 S ribosomal subunits is totally dependent on eIF-2 and stimulated approximately 3-fold by the 0.4 M KCI phosphocellulose fraction. Although activity observed at zero charge was 30% of that obtained at a charge of 1.0, 25% of total complex formation was observed in the absence of GTP. Poly(A,G,U) is not required for [35S]Met-tRNAf binding to 40 S ribosomal subunits under these conditions but does exert a slight stimulatory effect. The effect of adenylate energy charge on the extent of 40 S ribosomal initiation complex formation is shown in Fig. 3.
80 S Ribosomal Elongation Complex Assay To compare the effects of the adenylate energy charge on initiation complex formation to effects on elongation complex formation, GTPdependent binding of [3H]Phe-tRNA to 80 S ribosomes in the presence of varying adenylate energy charges was studied. The [3H]Phe-tRNA bound to ribosomes is determined by retention of the complex on Millipore filters as described by Hardesty et al. 14 and modified as outlined below.
588
INITIATION OF PROTEIN SYNTHESIS
"•v
I
I
I
I
0.2
0.4
0.6
0.8
[53]
100 ~ {:1:1 I--
8o-
60~
40-
I:L
20~L
0
1.0
ADENYLATE ENERGY CHARGE
Fro. 3. Effect of adenylate energy charge on 40 S ribosomal initiation and 80 S ribosomal elongation complex formation. Initiation complex was quantitated by sucrose gradient analysis and elongation complex by Millipore filter retention; 100%binding equals 0.42 and 1.18 pmol of [35S]Met-tRNAf(O) and [3H]Phe-tRNA(©), respectively. Adenylate pool was 2 mM, and guanylate pool was 0.1 mM. From G. M. Walton and G. N. Gill,Biochim. Biophys. Acta 447, 11 (1976). Reagents
Buffer T K M D Buffer T K M [3H]Phe-tRNA, 0 . 1 5 / x M GTP, 1 raM, sodium salt Poly(U), 10 mg/ml 80 S ribosomes, 100 A260 units/ml eEF-1, 4300 units/mg Procedure. To 20 ~1 of the adenylate energy charge reaction mixture the following components are added in a final reaction volume of 50/~1:5/xl of buffer T K M D ; 5/zl of 0.15 p~M [3H]Phe-tRNA; 5/xl of 1 m M GTP; 5 t~l of poly(U) (50/~g); 0.3 Az60 unit of 80 S ribosomes; and 1-3 units of e E F - I . After incubation at 37 ° for 30 min, the radioactivity bound is determined by Millipore filter retention as outlined for guanosine nucleotide complex formation. An assay blank is determined by omitting e E F - I . Figure 3 demonstrates the effect of the adenylate energy charge on both 40 S ribosomal initiation complex and 80 S ribosomal elongation complex formation. Preferential regulation of the initiation process is clearly evident as expected from the binding affinities ofeIF-2 and eEF- 1 for G T P and GDP (Table I). Remarks Adenine nucleotides do not directly effect formation of initiation and elongation complexes but exert indirect control through regulation of the
[53]
NUCLEOTIDE REGULATION OF PROTEIN SYNTHESIS
589
concentrations of GTP and GDP (Fig. 2C). In the absence of an adenylate pool, the GTP:GDP ratio determines the extent of initiation and elongation complex formation (Fig. 4). The broken line in Fig. 4 indicates the extent of 80 S elongation complex formation with GTP alone and demonstrates the GTPase activity of the 80 S ribosome. Magnesium ion-dependent inhibition of ternary initiation complex formation in crude ribosomal extracts appears to be the result of magnesiumdependent GTPase activity, because inhibition is overcome by GTP regeneration from ATP, and no inhibition is observed with purified elF-2 when GDP-free GTP is used. Further, the affinity of elF-2 for GTP or GDP was not appreciably affected by the presence or the absence of exogenous magnesium. 4 When a crude ribosomal extract is used as a source of initiation factors, the response to the adenylate energy is the same as that observed in the reconstructed system with purified components, e.g., 50% inhibition of ternary initiation complex formation at a charge of 0.85. ~ Because of the high affinity ofelF-2 for GDP, relative to GTP, minimal GTP hydrolysis gives rise to inhibitory levels of GDP. Elongation complex formation is less responsive to energy charge owing to the greater affinity of eEF-I for GTP, although some control is apparent with 50% inhibition observed at a charge of 0.60. In the presence of high adenylate energy charge, adenylate kinase and nucleoside diphosphate kinase, GTP regeneration occurs at the expense of ATP, while GDP levels are minimal and ribosomal complex formation remains optimal. At extremely low charge levels, GTP regeneration cannot occur, and, besides GTPase activity, I
r~
I---
I
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I00
f/
~== 6o 4o
n~ 20 o
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i
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0.2
0.4
0.6
0.8
1.0
GTP GDP+GTP
FiG. 4. Effect of GTP mole fraction on ternary initiation complex and 80 S ribosomal elongation complex formation. Assays were by the Millipore filter procedure; 100% binding equals 1.04 and 2.15 pmol of [:~sS]Met-tRNAf (O) and [3H]Phe-tRNA (©), respectively. [~H]Phe-tRNA bound with GTP alone at concentrations used in mole fractions (0---©). Guanylate pool was 0.1 mM. From G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 447, 11 (1976).
590
INITIATION OF PROTEIN SYNTHESIS
[53]
energy in the guanylate pool is depleted by the greater adenylate pool through AMP:GTP transphosphorylation catalyzed by adenylate kinase. In vivo levels of nucleotides suggest that normally energy charge levels in the cell are in the range of 0.8-0.9, '''23 and the adenylate pool exceeds the guanylate pool by 10- to 20-fold. '4''5 As shown in Fig. 3, initiation complex formation is most sensitive to changes in the adenylate energy charge in the range found within the cell. A sensitive regulation of protein synthesis thus results from small changes in energy availability and links the overall rate of protein synthesis to the metabolic state of the cell.
~z A. 2:3 T. z4 A. z5 C.
G. C h a p m a n , L. Fall, and D. E. Atkinson, J. Bacteriol. 108, 1072 (1971). R. Live and E. K a m i n s k a s , J. Biol. Chem. 250, 1786 (1975). S. Bagnara and L. R. Finch, Eur. J. Biochem. 41,421 (1974). Colby and G. Edlin, Biochemistry. 9, 917 (1970).
