[35] Binding of proteins to mRNA

392

INITIATION

OF PROTEIN

SYNTHESIS

[35]

mM creatine phosphate, 1.6 mM dithiothreitol, 20 mM HEPES, pH 7.8, 40 /xM spermine, 2 mM magnesium acetate, 144 mM KC1, 10 /zM of 19 unlabeled amino acids, a labeled amino acid, 0.8 mM ATP, 20/zM GTP, and buffer or eIF-2 to be assayed. Stimulation of Translation in the Presence of dsRNA

Principle In the presence of low concentrations of dsRNA, initiation of protein synthesis in reticulocyte lysates containing heroin is blocked after a brief delay. 2 The addition of eIF-2 is sufficient to overcome the block completely, 4,5 and causes protein synthesis to continue, the extent of stimulation being dependent on the amount of added eIF-2, up to a saturation level.

Procedure The procedure is identical to the one described in the preceding section, except that the iysate is made 50/xM in hemin before its addition to the reaction mixture, and dsRNA is added to a final concentration of 2-10 ng/ml. In this case, too, the sensitivity of the assay can be increased by employing a gel-filtered lysate supplemented with heroin.

[35] B i n d i n g o f P r o t e i n s to m R N A ~

By MICHAEL G. ROSENFELD and ALICE BARRIEUX Characterization of proteins involved in the process of mRNA translation has classically followed the demonstration that the partially purified protein functions in the process of initiation complex formation, elongation, or termination. An alternative method for the identification and characterization of proteins that may play a role in translation of natural mRNAs is to purify proteins with a high affinity for mRNAs. In this chapter, methods for purification and characterization of those mRNA binding proteins distinct from factors thus far identified in mRNA insertion into the initiation complex are described.

Reagents [5,6-3H]Uridine, 38 Ci/mmol (New England Nuclear) Rabbit liver methionyl-tRNAi (initiator methionine-tRNA, 95% pure, Investigations from the authors' laboratory were supported by American Cancer Society Grant No. BC-169C.

METHODS IN ENZYMOLOGY, VOL. LX

Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. 1SBN 0 12-18t960-4

[35]

BINDING OF PROTEINS TO m R N A

393

from Biogenics Research Co.) prepared according to Yang and Novelli" Stripped rabbit liver tRNA (General Biochemicals), dissolved in 1% sodium dodecyl sulfate (SDS), extracted with phenol 2 times, precipitated with ethanol 2 times, and redissolved in sterile water prior to use Pancreatic ribonuclease (Worthington) Formamide, 99% (Mallinckrodt, Inc.) stirred with mixed-bed resin AG501-X8(D) (Bio-Rad, reagent grade) and filtered prior to use F-10 medium (Gibco) RNase-free sucrose (Bio-Rad) Polyethylene glycol, 15,000-20,000 molecular weight (J. T. Baker) Guanosine triphosphate, guanosine diphosphate, adenosine triphosphate (P-L Biochemicals) Polyriboguanylic acid, polyriboadenylic acid, polyribouridylic acid, and polyribocytidylic acid (Miles Laboratories) were subjected to rate-zonal sedimentation, and the material migrating at 4-5 S was collected by precipitation in 66% ethanol and dissolved in sterile water prior to use Nucleoside diphosphate kinase (Boehringer Mannheim Corp.) Oligo(dT)-cellulose (T:~) (Collaborative Research) Phosphocellulose (Whatman, P- 11) DEAE-cellulose (Whatman, DE-52) Hydroxyapatite (Hypatite C, Ciarkson Chemical) (Ethylenedinitrilo)tetraacetic acid, disodium salt (Mallinckrodt), used as an 0.1 M stock solution, neutralized with NaOH to pH7 Ammonium sulfate (ultrapure, Schwarz/Mann) Nitrocellulose filters (0.45 p~m, Millipore) Liquifluor, 40 ml mixed with 960 ml toluene (New England Nuclear) All other chemicals used were reagent grade

Buffers Buffer A: 10 mM triethanolamine, pH 7.6, 10 mM KC1, 1 mM MgCI2 Buffer B: 4 M sodium acetate, pH 5, 10 mM EDTA Buffer C: 10 mM Tris.HCl, pH 8, 300 mM KCI, 0.5% sodium Nlaurylsarcosinate Buffer D: 10 mM Tris.HC1, pH 7.6, 30 mM KC1 Buffer E: 10 mM Tris.HC1, pH 7.6, 0.25 M sucrose, 1 mM dithiothreitol, 0.1 mM EDTA Buffer F: 10 mM Tris.HCi, pH 7.6, 100 mM KCI, 10% glycerol, 1 mM dithiothreitol, 0.1 mM EDTA W. K. Yang and G. D. Novelli, this series, Vol. 20 [5].

394

INITIATION OF PROTEIN SYNTHESIS

[35]

Buffer G: 10 m M potassium phosphate, pH 7.5, 10% glycerol, 1 m M dithiothreitol

Procedures All manipulations are performed at 4 °, unless otherwise stated. All glassware is heated for 24 hr to 180° prior to use; buffers are autoclaved after preparation, except that dithiothreitol is added after autoclaved buffers are chilled to 4 ° . Preparation ofmRNA. Ehrlich ascites tumor cells are inoculated into the peritoneal cavity of Swiss mice; 7 days later the cells are collected, centrifuged at 1000 g, and suspended at a concentration of 10~ cells/ml in 200 ml of F- 10 medium containing 15 m M H E P E S , pH 7.4, and 10/zCi/ml of [3H]uridine. After incubation at 37 ° for 16 hr, cells are harvested by centrifugation and washed twice with 50 ml of 0.9% NaC1. The cells are lysed in 10 ml of buffer A by gentle hand homogenization and the homogenate is centrifuged at 3000 g for 10 min. Total cytoplasmic RNA is prepared by adjusting the supernatant to 1% SDS and extracting with p h e n o l . c h l o r o f o r m . i s o p e n t a n o l (50.50.1, v/v/v) at room temperature until the phenol-water interphase is clear of protein precipitate. One-tenth volume of buffer B is added to the aqueous phase, and RNA is precipitated by the addition of 2.5 volumes of ethanol and storage for 18 hr at - 2 0 °. RNA is pelleted by centrifugation at 20,000g for 20 min and resuspended in 1 mi of buffer C. The RNA solution is applied at room temperature to an oligo(dT)-cellulose column prewashed with 10 ml of 50% formamide and 100 ml of water and equilibrated with 20 ml of buffer C. After sample application, the column is washed with buffer C until the radioactive content of the eluate is decreased to background level. Usually 30-35 ml of buffer C are required using a column containing 0.5 g of oligo(dT)-cellulose. Poly(A)-rich RNA is eluted with 10 m M Tris .HCI, pH 8.0; 0.5-ml fractions are collected, RNA elutes in fractions 3, 4, and 5. The three fractions are pooled and adjusted to contain 300 m M KCI; oligo(dT) chromatography is repeated a second time. Fraction 4, which contains 80% of the radiolabeled poly(A)-rich RNA, is stored in 50-pJ aliquots at - 8 0 °. Unlabeled poly(A)rich RNA is prepared from rabbit reticulocytes and Ehrlich ascites tumor cells by the same procedure. RNA not retained on oligo(dT)-cellulose is heated to 90 ° and applied to a 5 to 20% linear sucrose gradient containing buffer D and centrifuged at 25 ° for 2.5 hr at 45,000 rpm (200,000 g), using a Beckman SW 56 rotor, to separate 4-5 S, 18 S, and 28 S ribosomal RNA. When homogeneous labeled RNA is required, [:~H]poly(A)-rich RNA is dissolved in 98% formatnide, heated to 90 ° for I min, applied to a linear 5% to 20% sucrose density gradient containing 98% formamide, 4 M urea, and centrifuged for 40 hr at 45,000 rpm (200,000 g) using a Beckman SW 56

[35]

BINDING OR PROTEINS TO m R N A

395

rotor. The gradient is collected from the bottom of the tube and each fraction is diluted with 10 volumes of sterile water; one-tenth volume of buffer B is added, and the RNA is precipitated by the addition of 2.5 volume of ethanol and storage for 18 hr at --20 °. Reticulocyte 4 S, 18 S, and 28 S RNAs centrifuged in a separate gradient are used as markers. Binding of [3H]mRNA to Protein. [3H]mRNA (10-20 p,g/ml), the desired amount of unlabeled RNA species in 5/zl of H20, and the protein to be assayed in 5/zl of buffer F are sequentially added to 35/zl of buffer D and the reaction is allowed to proceed at 4 ° for 10 rain. The reaction is terminated by the addition of 1 ml of ice-cold buffer D. Binding is measured by slow filtration through a nitrocellulose filter, and washing of the filter with an additional 30 ml of ice-cold buffer D. The filters are dried and counted in 10 ml of Liquifluor-toluene. The binding of [:~H]poly(A)-rich RNA to Millipore filters in the absence of added protein represents <0.3% of added RNA, and is subtracted from each experimental Tesult. Purification of Three m R N A Binding Proteins

Preparation of 0.5 M KCI Ribosomal Eluate and Phosphocellulose Chromatography. The 0.5M KCI ribosomal eluate is prepared as described by Crystal et al. :~The 60% ammonium sulfate precipitate is collected by centrifugation at 15,000g for 15 rain, resuspended in 5 ml of buffer F, and dialyzed for 18 hr against 1 liter of buffer F. The solution is then centrifuged at 5000g for 10 rain to remove insoluble material, and applied to a 1.5 cm × 5 cm phosphocellulose column previously equilibrated with buffer F. After sample application, the column is washed extensively with buffer F until no further material elutes; sequential batch elutions are performed using buffer F containing 0.2, 0.3, 0.4, 0.5, 0.6, and 1M KC1, with extensive washing of the column (60-100 ml) between each step. Each fraction is concentrated by dialysis against buffer F containing 9% polyethylene glycol. Messenger RNA binding activity can be observed in each fraction. A. Purification of a Three-Subunit mRNA-Binding Protein Copurifying with GTP-Dependent Met-tRNAiBinding Protein. After phosphocellulose chromatography, the 0.5 M KCI fraction is dialyzed against buffer F and applied to a DEAE-cellulose (0.5 X 5 cm) previously equilibrated with the same buffer; the column is washed with 40 ml of buffer F. Met-tRNAi and mRNA binding activities are eluted using buffer F containing 0.2 M KC1; 0.94 pmol of Met-tRNAi and 1.28 pmol of poly(A)-rich mRNA are bound per microgram of protein. As shown by SDS-polyacrylamide gel electrophoresis (Fig. IA) this fraction is greatly enriched for eIF-2. The three major bands of 52,000, 48,000, and 37,000 apparent molecular weights :~R. G. Crystal, N. A. Elson, and W. F. Anderson, this series, Vol. 30 [13].

396

INITIATION OF PROTEIN SYNTHESIS

[35]

A

BSABgalBSA-

OVBSA-

OV0V-

TI-

Fro. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of mRNA-binding proteins, elF-2 (A), the 2-subunit protein (B), or the 8-subunit protein (C) were analyzed using gels containing either 8% or 10% polyacrylamide. Standards are /3-galactosidase (/3gal), bovine serum albumin (BSA), ovalbumin (OV), and soybean trypsin inhibitor (TI).

represent the three putative subunits of elF-2. As reported previously, Met-tRNAi binding requires GTP and is inhibited by GDP. 4 Neither GTP nor GDP have any effect on m R N A binding, elF-2 itself accounts for more than 50% of the mRNA binding activity of this fraction as determined by further purification using isoelectrofocusing. The 48,000 subunit of elF-2 is responsible for mRNA and Met-tRNAi binding. 5 B. Purification o f a Two-Subunit mRNA-Binding Protein. The concentrated 0.2 M KC1 phosphocellulose eluate is diluted with 20 volumes of buffer G and applied to a 1 cm × 5 cm hydroxyapatite column previously 4 G. M. Walton and G. N. Gill, Biochim. Biophys. Acta 390, 231 (1976). .3 A. Barrieux and M. G. Rosenfeld, J. Biol, Chem. 252, 3843 (1977).

[35]

BINDING OF PROTEINS TO m R N A

397

equilibrated with the same buffer. Batch elution is performed using buffer G containing 0.1,0.2, 0.4, and 0.6 M potassium phosphate; mRNA binding activity is resolved into several components, most activity eluting in the 0.4 M potassium phosphate fraction. The material eluting in this fraction is extensively dialyzed against buffer F and applied to a 0.5 × 10 cm DEAEcellulose column previously equilibrated with the same buffer. Following extensive washing with buffer F, batch elution is performed using buffer F containing 0.2 M KC1. The protein recovered in this fraction binds 0.7-0.8 /zg of mRNA per microgram of protein and contains two prominent proteins migrating with apparent molecular weights of 56,000 and 61,000 on SDS, polyacrylamide gel electrophoresis (Fig. 1B). The two proteins comigrate in isoelectrofocusing gels, the complex sediments with an s,,0.wof 5.6-5.8S; and it exhibits an apparent molecular weight of 100,000 by gel filtration, suggesting that the two bands observed using SDS-polyacrylamide gel electrophoresis are subunits of a single proteinf C. Purification of an 8-Subunit mRNA-Binding Protein. A multisubunit protein present in the 0.5 M KCl ribosomal eluate can be separated from the multiple-subunit protein now referred to as elF-3, r by two-dimensional gel electrophoresis of the 0.4 M KCI phosphocellulose eluate. The protein is most conveniently purified by stirring the ribosomal pellet with 26 ml of buffer E containing 0.15 M KC1. The suspension is then centrifuged as described for purification of elF-2. More than 90% of this protein is eluted from ribosomes at this KC1 concentration. The 0.15 M KCl eluate is adjusted to 40% saturation with respect to ammonium sulfate by the addition of 5.6 g of ammonium sulfate and allowed to stir for 1 hr at 4°; the pellet is then collected by centrifugation for 15 min at 15,000 g and dissolved in 5 ml of buffer F. The solution is dialyzed extensively against buffer F and phosphocellulose chromatography is performed exactly as described for the total 0.5 M KCI ribosomal eluate. The material eluting at 0.4 M KC1 is dialyzed and concentrated to a 300-/zl volume against buffer F containing 9% polyethylene glycol, diluted with 500/zl of buffer F containing no KCI nor glycerol, and 200-/A aliquots are applied to 4-ml linear 5 to 20% sucrose density gradients containing buffer F without glycerol. After centrifugation at 45,000 rpm (200,000 g) for 2.5 hr using a Beckman SW 56 rotor, fractions are collected from the bottom and those fractions migrating at 16-18 are pooled and represent an apparently homogeneous preparation of the multisubunit protein. Analysis on SDS-polyacrylamide gel electrophoresis reveals 8 subunits of 38,000, 56,000, 72,000, 97,000, 110,000, 140,000, 160,000, and 170,000 apparent molecular weights; the complex appears to contain 2 mol of the MW 72,000 subunit per mole of other subunits. The M. G. Rosenfeld and A. Barrieux, Biochemistry 16, 514 (1977). 7 I. C. Sundkvist and T. Staehelin, J. Mol. Biol. 99, 401 (1975).

398

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

[35]

multiple subunit protein is excluded by Sephadex G-200 gel filtration and migrates as one complex when it is analyzed on nondenaturing isoelectrofocusing gels. The purified protein preparation binds approximately 0.5 /zg of {3H]poly(A)-rich mRNA per microgram of protein. Properties of the m R N A Binding Assay

Validity of the mRNA Binding Assay. The validity of the nitrocellulose binding assay is confirmed by sedimentation analysis. Two methods are used: In method 1, 0.5-1 /zg of 10-11 S [:~H]mRNA, prepared from Ehrlich ascites tumor cells, is heated to 90° for 3 min and then incubated for 10 min at 4° in the presence of an excess amount of mRNA-binding protein. The migrations of [:~H]mRNA alone and [:~H]mRNA .protein complex are then compared by applying to a 4-ml linear 5 to 20% sucrose density gradient containing 10 mM Tris .HC1, pH 7.6, and l0 mM KCI. The gradients are centrifuged at 4° at 45,000 rpm (200,000g) for 200 min using a Beckman SW 56 rotor. Fractions are collected from the bottom, adjusted to 10% trichloroacetic acid, filtered through glass-fiber filters (Whatman GF/C), washed with 20 ml of 5% trichloroacetic acid and 2 ml of acetone, dried, and counted in 5 ml of Liquifluor-toluene. In method 2, an identical procedure is performed except that, instead of [:~H]mRNA, unlabeled 9 S rabbit reticulocyte mRNA is added in excess to the binding capacity of the binding protein; analysis of the gradient is performed by SDS-polyacrylamide gel electrophoresis of each fraction. Using the first method, a shift in sedimentation of [:~H]mRNA from I 1 S to 16 S is demonstrated following its binding to elF-2. 8 Using the second method, a shift from 17 S to 24 S is observed in sedimentation of the 8-subunit protein. Specificity of mRNA Binding. The relative affinity of each protein for mRNA compared to other natural and synthetic RNAs is assessed by determining the ability of various unlabeled species of RNA to compete with [3H]poly(A)-rich RNA. In the experiment shown in Fig, 2, binding of [:~H]mRNA to the two-subunit protein is measured in the presence of other RNA species by the Millipore filter assay. Most RNA species are 90- to 250fold less effective than mRNA at competing for binding. The binding of mRNA by elF-2 is relatively less specific because most RNA species are only 10- to 100-fold less effective than mRNA. Polyriboguanylic acid, however, is more effective than mRNA at competing for binding by either protein. In contrast, no such specificity of mRNA is observed with respect 8 A. Barrieux and M. G. Rosenfeld, J. Biol. Chem. 252, 392 (1977).

[35]

BINDING OF PROTEINS TO m R N A

L o

i

399

i

2ooo

,00t\\ t \\

:

,oo

0

i

i

I0

I00

RNA ADDED (/~g)

FIG. 2. Competition of mRNA binding by the two-subunit protein by natural and synthetic RNAs. Unlabeled RNA added was: (A) poly(G), (B) poly(A)-rich mRNA from Ehrlich ascites tumor cells, (C) poly(A)-rich mRNA from rabbit reticulocytes, (D) tRNA, (E) 18 S ribosomal RNA, (F) poly(A), (G) poly(A).poly(U), (H) poly(AUG), (I) poly(U). From M. G. Rosenfeld and A. Barrieux, Biochemistry 16, 514 (1977).

to the 8-subunit protein. Of these three proteins, only elF-2 binds significant amounts of [3'~S]Met-tRNAi; mRNA appears to compete at equimolar concentrations with [3~S]Met-tRNA1 for binding of elF-2. Subunit Binding ofmRNA. Isoelectrofocusing techniques, described in detail in a previous chapter, 9 provide an opportunity to determine whether mRNA binding introduces any marked structural alterations in a polymeric protein; such a conformational alteration is produced by binding of mRNA to elF-2. The protein in buffer F is incubated with varying concentrations of rabbit reticulocyte mRNA for 10 min at 4° and analyzed by twodimensional gel electrophoresis. The first dimension, isoelectrofocusing, is performed under nondenaturing conditions. '~In the absence of mRNA, the three subunits of elF-2 comigrate (Fig. 3A); in the presence of mRNA subunit dissociation is observed. Most of the MW 37,000 subunit isoelectrofocuses at a pI identical to that of the isolated subunit, while the MW 52,000 and 48,000 subunits comigrate at the basic end of the isoelectrofocusing gel (Fig. 3B). This mRNA-induced modification of elF-2 is dependent upon the concentration of added mRNA and upon intact mRNA; it is abolished by preincubation of mRNA with pancreatic ribonuclease (2 p~g/mi)for 15 min at 30°. When Met-tRNAi is bound to elF-2, under conditions described in a previous chapter, '~elF-2 migrates at a more acidic pI than in the absence of Met-tRNA~, and no subunit dissociation is observed (Fig. 3C). A. Barrieux and M. G. Rosenfeld, this volume [22].

400

INITIATION OF PROTEIN SYNTHESIS

[35]

IEF ACIDIC

elF-2

BASIC

A

52,000_ 48,000 37,000--

e l F - 2 + mRNA

52,000-48,000

I

37,000--

elF-2 + Met-tRNA i

52,000_ 48,000--

;~

37,000--

FIG. 3. Two-dimensional gel electrophoresis of elF-2, elF-2 was incubated alone or in the presence of ~-fold molar excess of rabbit reticulocyte poly(A)-ricb mRNA or Met-tRNAi, and then subjected to two-dimensional gel electrophoresis. A. Barrieux and M. G. Rosenfeld, this volume [22].

[36]

MESSENGER

RNA

BINDING

TO

RIBOSOMES

401

Comments The purification of three proteins that have a high affinity for mRNA has been described in this article. One of these proteins is initiation factor 2. mRNA has an affinity comparable to that of Met-tRNA~ and competes with Met-tRNAi for binding to this factor; both mRNA and Met-tRNAi bind to the MW 48,000 subunit? These observations suggest several possible functional consequences: (1) the ratio of Met-tRNAi and mRNA in the cell could determine which species binds eIF-2, and, therefore, whether the (40 S • eIF-2 • GTP • Met-tRNA~) initiation complex will be formed, and/or (2) mRNA binding to eIF-2 could play a role in its dissociation and release from the ribosome, and hence be required for the recycling of eIF-2. Many properties of the MW 48,000 subunit ofeIF-2 are identical to those of one of the major proteins associated with cytoplasmic mRNA. 6 These two proteins could either be identical, in which case a recycling of the MW 48,000 subunit of elF-2 with mRNA.protein particles might occur; or they are different proteins with similar properties. Bol~h eIF-2 and the 2-subunit mRNA-binding protein described here exhibit an apparent specificity for mRNA; in contrast the 8-subunit protein binds all species of RNA tested with equal affinity. Binding at 4° is rapid, being complete in 30-60 sec for all three proteins. The apparent K ~ T M for the 2- and 8-subunit proteins is between 10-~ and 10-~° M. Any biological role for these proteins in mRNA translation remains to be demonstrated.

[36] B i n d i n g o f I o d i n e - L a b e l e d m R N A to 40 S R i b o s o m a l S u b u n i t s By A. B. HENDERSON, G. KRAMER, and B. HARDESTY Many early attempts to study the individual reactions in the process of polypeptide chain initiation were limited by the availability of labeled mRNA. Necessary requirements are that the mRNA retain its biological activity and be easily detectable in very small amounts. Here, we describe a procedure for preparation of biologically active v'5I-mRNA of high purity and specific radioactivity; we also present procedures for the assay of binding of this radioactively labeled mRNA to Artemia salina ribosomes and 40 S ribosomal subunits. Buffers and Solutions All chemicals used in the following procedures are of reagent grade. Buffers are prepared and the pH is measured at 25°. METHODS IN ENZYMOLOGY. VOL. LX

Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4