the journal of chemistry vol ,267, no. 3, pp. 1554-1562 ... · the journal of biological chemistry...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol ,267, No. 3, Issue of January 25, pp. 1554-1562,1992 Printed in U. S. A.

Analysis of 40 S and 80 S Complexes with mRNA as Measured by Sucrose Density Gradients and Primer Extension Inhibition*

(Received for publication, August 29, 1991)

Donald D. Anthony and William C. Merrick From the Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleueland, Ohio 44106

The technique of primer extension inhibition has been adapted to analyze the eukaryotic ribosome- mRNA interaction. Formation of the ribosome-mRNA complex was performed in a nuclease-treated rabbit reticulocyte lysate. Before primer extension analysis, however, the complex is isolated by sucrose gradient centrifugation. Both 80 S- and 40 S-mRNA complexes can be individually analyzed because of this isolation step. 80 S ribosomes and 40 S ribosomal subunits could be localized at the initiation codon by a number of independent means where all complexes were formed in a manner consistent with the current understanding of the initiation pathway for translation in eukaryotes. Complexes were also isolated with the aid of the antibiotic edeine, where the 40 S ribosomal subunit was not located at the initiation codon, but 5‘ to the initiation codon. This extension inhibition assay was used to complement studies regarding the ATP dependence of the 40 S-mRNA interacting initiation steps that in- volve the mammalian RNA-interacting initiation factors eIF-4A, -4B, and -4F. A strong requirement for ATP was observed for 40 S-mRNA complex formation. A factor-mediated stimulation of complex formation by a combination of eIF-4A, -4B, and -4F was observed, and was one which required the presence of ATP. This factor-mediated ATP-dependent stimulation of complex formation was significantly inhibited by preincubating eIF-4A with the ATP analog 5‘-p- fluorosulfonylbenzoyl adenosine. Finally, all complexes accumulated to a significant degree were analyzed by the primer extension assay. It was found that the 40 S ribosomal subunit was positioned at the initiation codon for all variations tested.

Extension inhibition analysis has been previously used to study the prokaryotic ribosome-mRNA interaction and to better characterize the role of prokaryotic initiation factors in achieving a ribosome positioned on an mRNA at the initiation codon (1-3). This technique involves primer extension from an oligodeoxynucleotide, which is hybridized 3’ to the initiation codon of an mRNA. Extension to the 5’ termi- nus of the mRNA results in a run off product. Inhibition of primer extension, by a ribosome stably complexed to an mRNA, can lead to a shorter extension product, indicative of the location of the ribosome. The collision event between the reverse transcriptase enzyme and the ribosome in prokaryotes yields an extension product which corresponds to the nucleo-

* This work was supported by National Institutes of Health Grant GM-26796 and National Institutes of Health Training Grant T32- GM-07520. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

tide 15 bases 3’ to the A in the initiation codon (1). An adaptation of the extension inhibition technique has

been developed in order to analyze the eukaryotic ribosome- mRNA interaction. The complex is formed under conditions compatible with initiation. Before extension inhibition analysis, however, the ribosome-mRNA complex is isolated by sucrose gradient centrifugation. As a result, 80 S-mRNA and 40 S-mRNA complexes can be individually analyzed. Ribo- some-mRNA complexes were formed in rabbit reticulocyte lysate with the aid of the elongation inhibitor anisomycin (4), and the 80 S complex formation inhibitor GMP-PNP’ ( 5 ) . A 40 S-mRNA complex formed in the presence of the initiation site recognition inhibitor, edeine (6), was also analyzed. A controlled set of parameters ensured that authentic initiation complexes were represented by this assay. All complexes were formed in the expected cap-dependent fashion. Complexes were also formed in a manner consistent with the predicted initiation path scheme (40 S-mRNA complexes formed prior to 80 S-mRNA complexes). Finally, in addition to analyzing translation inhibitor-dependent complexes, 40 S- and 80 S- mRNA complexes that can be isolated over a short time range in the absence of inhibitors were also analyzed. The primer extension inhibition assay was subsequently used to monitor the location of the 40 S ribosomal subunit during translation initiation events.

The scanning hypothesis, originally proposed by Kozak (7), serves as a two-step model for eukaryotic translation initiation. In the first step the 40 S ribosomal subunit interacts with the 5‘ end of the mRNA. In the second step the 40 S subunit moves in an ATP-dependent manner down the mRNA in a 5’ + 3’ direction, “scanning” for the proper initiation codon, where the 60 S ribosomal subunit joins the 40 S subunit to form a translationally competent 80 S ribosome. Ribosomes can be loaded onto the 5’ end region of an mRNA in the presence of the antibiotic edeine, and can be shown to migrate to the 3‘ end of the mRNA in an ATP- dependent fashion (8). Evidence from yeast genetic experiments has also strongly supported the hypothesis of scanning (9, 10).

The requirement for initiation factors to achieve a 40 S ribosomal subunit or an 80 S ribosome complexed with an mRNA has been studied (11, 12). The initiation factors eIF- 2, eIF-3, eIF-4A, eIF-4B, eIF-4F, ATP, and Met-tRNAi are required to achieve a 40 S ribosomal subunit complexed with the mRNA stable enough to isolate by sucrose gradient centrifugation. From model activity assays with these initiation factors and RNA, in the absence of ribosomes, a sequence of events for the interaction of initiation factors with the mRNA

’ The abbreviations used are: GMP-PNP, guanyl-5’-yl imidodi- phosphate; eIF, eukaryotic initiation factor; FSBA, 5‘-pfluorosulfon- ylbenzoyladenosine; FSBG, 5’-p-fluorosulfonylbenzoylguanosine; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid Met- tRNA,, initiator methionyl tRNA species.

1554

Primer Extension Inhibition with Eukaryotic Ribosomes 1555

has been proposed where eIF-4F is the first initiation factor to bind the mRNA (at the cap structure) in an ATP independent fashion; eIF-4B then binds to eIF-4F, if in fact it was not already bound to eIF-4F prior to the eIF-4F. mRNA binding step; and finally, eIF-4A binds to the eIF-4F.eIF-4B. mRNA complex and functions in an ATP dependent manner to facilitate unwinding of the mRNA necessary for 40 S subunit association and/or scanning (13). However, the particular site of action of all of these components can only be inferred from these model activity assays performed in the absence of the ribosome. Specifically, the particular role of eIF-4A, eIF-4B, and eIF-4F, as well as that of ATP during initiation is not known. While these mRNA interacting factors are capable of secondary structure unwinding activity (14, 15), the relationship of this unwinding to initiation is not yet clear.

An ATP-depleted lysate has been utilized in conjunction with the primer extension inhibition assay to assess the ATP dependence of the 40 S-mRNA interacting initiation steps. The ATP dependence of the mRNA interacting initiation factors, eIF-4A, eIF-4B, and eIF-4F, was of primary concern. A strong ATP requirement for 40 S-mRNA complex formation was seen. Any significant quantity of complex that could be detected before the ATP was completely depleted from the system indicated that the 40 S ribosomal subunit was positioned at the initiation codon. Also, the factor-mediated stimulation of ribosome-mRNA complex formation by the combination of eIF-4A, eIF-4B, and eIF-4F requires the presence of ATP. Furthermore, this factor-mediated stimulation of ribosome-mRNA complex formation was sensitive to the pretreatment of eIF-4A, but not eIF-4B or eIF-4F, with the ATP analog FSBA, supplying additional evidence that this factor likely accounts for the ATP requirement of ribosome binding and/or scanning. The 40 S ribosomal subunit, in all complexes isolated, was located at the initiation codon of the mRNA, eliminating the possibility that the factor-mediated stimulation led only to a complex of the 40 S ribosomal subunit with the 5' leader of the mRNA.

MATERIALS AND METHODS

Purification of mRNA Binding Initiation Factors and 9 S Globin RNA-Purification of eIF-4A, eIF-4B, and eIF-4F to greater than 90% homogeneity was carried out as previously described (16-19). All factors were tested for activity, as described previously, in a reconstituted globin synthesis assay (16, 18, 20).

Rabbit 9 S globin RNA was obtained from the reticulocyte lysate as described previously (17). Product RNA was tested for transla- tional activity by hot trichloroacetic acid precipitable radioactivity in a micrococcal nuclease-treated reticulocyte lysate assay. Aliquots of reactions were also subjected to sodium dodecyl sulfate-polyacryl- amide gel electrophoresis on 20% acrylamide gels, followed by auto- radiography, in order to analyze the size and integrity of the encoded globin protein products.

Oligodeoxynucleotide Kinase Reaction-Oligodeoxynucleotides were reacted with T4 polynucleotide kinase (Boerhinger Mannheim Biochemicals) and [y-32P]ATP (Amersham Corp.) of specific activity greater than 5000 Ci/mmol. A reaction (10 pl) contained 10 pmol of oligodeoxynucleotide, 12 pmol of [y-32P]ATP, 50 mM Tris-HC1, pH 7.5, 10 mM MgC12, 5 mM dithiothreitol, and 3 units of T4 polynucleotide kinase. The mixture was incubated 30 min at 30 "C. The volume was brought to 100 p1 with 10 mM Tris-HC1, pH 7.5, and 0.1 mM EDTA. This mixture was passed over a 1-ml Sephadex G-50m (Phar- macia Fine Chemicals) spin column.

Hybridization of a 32P End-labeled Oligodeoxynucleotide to an mRNA-The mixture of end-labeled oligodeoxynucleotide and mRNA contained 30 mM NaCl, 25 mM Tris-HC1, pH 8.3, 10 mM dithiothreitol and oligodeoxynucleotide-mRNA. The mixture was heated to 100 "C for 1 min and then allowed to cool to room temper- ature on the bench top. For each translation reaction roughly 1 pg of purified 9 S globin RNA and 1.5 pCi of kinased oligodeoxynucleotide were used. Oligodeoxynucleotides complementary to a region 60 nu-

cleotides 3' to the A in the initiation codon of @-globin (oligodeoxynucleotide: 5'-TCACCACCAACTTCTTCCAC-3') and a-globin (oligodeoxynucleotide: 5'-GCGCCATACTCGCCACCGTG-3') were synthesized on an Applied Biosystems Inc. model 380B DNA synthe- sizer in the Case Western Reserve University Molecular Biology Laboratory.

Complex Formation and Isolation-Micrococcal nuclease-treated rabbit reticulocyte lysate (Promega) was the source of ribosomes, initiation factors, and a number of other components necessary for initiation of protein synthesis. The ribosome-mRNA complex, localized at the initiation codon, was trapped by the use of late initiation (GMP-PNP) or early elongation (anisomycin) inhibitors. In the case of the elongation inhibitor anisomycin (Sigma), a preincubation of the antibiotic with the lysate was performed (5 min a t 30 "C) in order to bind the antibiotic to all ribosomes before any elongation steps of translation can occur. A typical reaction (150 pl) consists of nuclease- treated rabbit reticulocyte lysate (50 pl), 15 mM Hepes-KOH, pH 7.5, 100 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 0.1 mM anisomycin, or 1 mM GMP-PNP (Sigma), and a 32P-end-labeled oligodeoxynucleotide-mRNA hybrid. The final reaction mixture (150 pl) was incubated for various amounts of time (0.5-10 min) at 30 "C in the presence or absence of 0.2 mM m7GTP. After incubation, the reactions were brought to 4 "C on ice, and then subjected to centrifugation with a SW 40 rotor (Beckman) for 3.75 h a t 4 "C in 10-35% linear sucrose gradients containing 60 mM NaCl, 6 mM MgC12, 50 mM Tris-HCI, pH 8.4, and 10 mM dithiothreitol. The gradients were fractionated (0.7- ml fractions: 17 fractions/gradient) and 0.3 ml of each fraction was counted via liquid scintillation spectrometry. A graph of radioactivity uersus sucrose gradient fraction number was plotted to determine the S value and quantity of [32P]oligodeoxynucleotide-mRNA present at each S value.

Formation of ribosome-mRNA complexes in edeine-treated lysate was also performed. The antibiotic edeine was supplied by Dr. Rose- mary Jagus (Center of Marine Biotechnology, University of Mary- land, Baltimore, MD). Treatment of lysate with 2 p~ edeine results in over 90% inhibition of translation. A pretreatment of lysate with the edeine for 2 min at 30 "C was performed to ensure that the ribosome population was bound by the antibiotic before mRNA addition.

Analysis of Ribosome-mRNA Complexes by Primer Extension In- hibition-Aliquots (200 pl) of radioactive peak tubes in the 80 s, 40 S, or free RNA (9 S ) sucrose gradient regions were analyzed via primer extension inhibition with avian myeloblastosis virus reverse transcriptase (Molecular Genetic Resources). A reaction was initiated by addition of all 4 dNTPs to the 200-4 aliquot (0.2 mM final concentration) and 4 units of avian myeloblastosis virus reverse transcriptase. The reaction was incubated 30 min a t 37 "C. Reactions were then extracted with pheno1:chloroform (1:l) followed by ethanol precipitation. Pellets were solvated in sequencing gel buffer and subjected to 8% acrylamide sequencing gels. Extension reaction products were compared with a dideoxynucleotide sequence ladder obtained from the same 32P-end-labeled oligodeoxynucleotide primer and mRNA used for forming the ribosome-mRNA complex. For dideoxynucleotide sequencing reactions, 0.15 mM dideoxynucleotide was added to the reaction.

ATP Depletion of Lysate and FSBA Treatment of Initiation Fac- tors-ATP was depleted from the nuclease-treated rabbit reticulocyte lysate by the use of hexokinase and glucose, before incubation of the lysate with the oligodeoxynucleotide-mRNA hybrid. All salts and volumes were the same as those described under "Complex Formation and Isolation" except the reaction mixture was treated for 3 min a t 30 "C before the complex formation with 40 pg of yeast hexokinase (Boehringer Mannheim) and 3 mM glucose to completely deplete the lysate with respect to ATP. When ATP (as an ATP-MgZ' complex) was added back to the system, after the ATP depletion step, 20 mM lexose was included to compete with glucose for hexokinase.

Pretreatment of initiation factors with the ATP analog 5'-p- fluorosulfonylbenzoyladenosine (FSBA) was carried out as follows. All factors were incubated for 1 h at 30 "C. Those factors treated were incubated in the presence of 2 mM FSBA and 10% dimethylform- amide.

RESULTS AND DISCUSSION

Elongation Arrested Complexes and Initiation Arrested Complexes (60 S Subunit Joining Deficient)-Before initiation complexes were formed, an mRNA and a complementary

1556 Primer Extension Inhibition with Eukaryotic Ribosomes

oligodeoxynucleotide were chosen. Milligram quantities of 9 S globin RNA, composed of a-globin and @-globin mRNA, are available from rabbit reticulocyte lysate. Because of this avail- ability and the high level of efficiency with which these mRNAs are translated in nuclease-treated rabbit reticulocyte lysate (@-globin somewhat more so than a-globin), the 9 S globin RNA was chosen as the source of both a- and @-globin mRNA. Purification of this RNA was carried out as described under "Materials and Methods." In order to analyze a ribosome complexed to the initiation codon of the @-globin mRNA, a 20-mer oligodeoxynucleotide was designed to hy- bridize approximately 60 bases 3' to the AUG initiation codon. Similarly, a 20-mer oligodeoxynucleotide was designed for the a-globin mRNA. The distance of 60 bases 3' to the initiation codon is roughly the same as that used for the analysis of prokaryotic ribosome-mRNA complexes (1).

Ribosome-mRNA complexes were formed in a nuclease- treated rabbit reticulocyte lysate, as this is the most efficient eukaryotic cell-free protein synthesis system available, effect- ing translation at near in vivo rates (21, 22). In this way the authentic ribosome-mRNA complex could be monitored, as opposed to an artifactual event possibly occurring in a less translationally efficient reconstituted system. Also, to ensure that authentic initiation complexes were present, formation of 40 S-mRNA and 80 S-mRNA complexes localized at the initiation codon had to occur in the expected cap-dependent fashion. For this reason, sensitivity of complex formation to cap analog (m7GTP) addition was monitored.

Localization of the 40 S ribosomal subunit at the initiation codon was achieved by adding a 60 S ribosomal subunit joining inhibitor (GMP-PNP) (5) to the nuclease-treated rabbit reticulocyte lysate, before mRNA addition. Trapping of a later stage initiation complex/early elongation phase complex was achieved by preincubating the lysate with the elongation inhibitor anisomycin (4). These two inhibitors allowed independent means of trapping initiation complexes localized in roughly the same place on the mRNA. Because a concentration of 1 mM GMP-PNP or 0.1 mM anisomycin yielded over 90% inhibition of translation in rabbit reticulocyte lysate, these concentrations of inhibitor were used.

The 32P-end-labeled oligodeoxynucleotide was hybridized to the mRNA prior to incubation with the ribosome source, as was done in the prokaryotic experiments carried out by Hartz et al. (1). This enabled the use of efficient hybridization conditions not necessarily compatible with translation initiation. Also, this mRNA-[32P]oligodeoxynucleotide hybrid allowed detection of the mRNA throughout the ribosome- mRNA complex isolation procedure of sucrose gradient centrifugation, by liquid scintillation spectrometry.

It should be noted at this point that extension inhibition analysis of ribosome-mRNA complexes directly in lysate is not possible. Very little primer extension activity is noted when a reaction is attempted directly in lysate. This is not likely due to an RNase H activity present in the lysate, as a "P-labeled mRNA remains intact after an appropriate incubation in lysate when hybridized to an oligodeoxynucleotide. Also, the lack of primer extension activity is not likely due to a phosphatase activity, as no decrease in the 32P-end label attached to oligodeoxynucleotide is noted, and incorporation of [a-32P]dNTPs in the extension reaction doesn't allow the detection of previously undetected primer-extended products. Instead, this inhibition is more likely due to a direct inhibition of the extension reaction itself, or an activity capable of unwinding the oligodeoxynucleotide-mRNA hybrid. The latter explanation is favored (discussed below). In any case, an attempt to isolate the ribosome-mRNA complex by sucrose

gradient before extension inhibition analysis was made. As stated above, this isolation step also enabled the resolution of 40 S-mRNA complexes from 80 S-mRNA complexes. A schematic representation of complex formation, isolation, and characterization is given in Fig. 1.

The radioactivity profile of a typical sucrose gradient centrifugation isolation procedure performed on ribosome- mRNA (@-globin mRNA) complexes formed in the presence of 0.1 mM anisomycin is depicted in Fig. 2a. As seen in this figure, complex formation is cap-dependent because it is sensitive to the presence of 0.2 mM cap analog (m7GTP). Also, while a minor 40 S-mRNA complex can be seen, the primary ribosome-mRNA complex formed after a 10-min incubation at 30 "C in the anisomycin-treated lysate sediments at 80 S, indicating th.at this is an 80 S ribosome-mRNA complex. It is important to note that under these same incubation conditions, in the absence of anisomycin, no detectable ribosome- mRNA complex is seen (data not shown). In a cell lysate active in protein synthesis, it is likely that by 10 min at 30 "C the oligodeoxynucleotide-mRNA hybrid would be unwound by an elongating ribosome.

The anisomycin-dependent 80 S-mRNA and 40 S-mRNA extension inhibition patterns are represented in Fig. 2b. In- dividual sucrose gradient fraction aliquots were subjected to primer extension analysis by adding all 4 dNTPs (200 PM final concentration) and 4 units of avian myeloblastosis virus reverse transcriptase followed by a 30-min incubation at 37 "C. As can be seen in this figure, the extension inhibition patterns indicate extension stop sites that correspond to bases 15,16, and 17 3' to the A in the initiation codon for the minor 40 S-mRNA complex. The major 80 S-mRNA complex yields extension stop sites corresponding to bases 19 and 25-29 3' to the A in the initiation codon. The position of the 80 S- mRNA complex somewhat 3' of the 40 S-mRNA complex reflects a partial elongation movement before complete arrest of the ribosome on the mRNA is achieved. The possibility of

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FIG. 1. Schematic of formation, isolation, and characterization of ribosome-mRNA complexes. This figure represents the formation of a ribosome-mRNA complex, the subsequent isolation of this complex by sucrose gradient centrifugation, and the analysis of the specific 80 S-associated, 40 S-associated, or free 9 S sedimenting mRNA species by primer extension.


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FIG. 2. a, sucrose gradient profile of 32P-labeled oligodeoxynucleotide-mRNA incubated with anisomycin-treated rabbit reticulocyte lysate. A ribosome-mRNA (@-globin mRNA) complex was formed in anisomycin-treated (0.1 mM) rabbit reticulocyte lysate over an incubation for 10 min a t 30 “C as described under “Materials and Meth- ods.” The complex was subjected to sucrose gradient centrifugation on 10-35% linear sucrose gradients. A graph of radioactivity uersus sucrose gradient fraction number is represented in both the presence and absence of added m7GTP (0.2 mM). The position of sedimentation for 80 S ribosomes, 40 S ribosomal subunits, and free 9 S globin RNA are marked a t the top of the figure. b, extension inhibition analysis of 80 S-mRNA and 40 S-mRNA complexes obtained with anisomycin- treated rabbit reticulocyte lysate. Aliquots (200 pl) of peak tubes of radioactivity in the 80 S and 40 S gradient regions were analyzed by primer extension as described under “Materials and Methods.” Ex- tension reaction products from the 80 S and 40 S gradient regions are shown next to a dideoxynucleotide sequence ladder obtained with the same oligodeoxynucleotide primer and mRNA (@-globin mRNA) used for the complex. Extension in the absence of added dideoxynu- cleotides is also represented (+). The sequencing lanes are taken from a longer exposure of the same gel used for the other above represented lanes. The nucleotide on the mRNA that corresponds to the migration position of the extension inhibition product and its position 3’ to the A in the initiation codon is represented on the right. Below is a representation of the sequence of @-globin mRNA, the position of the initiation codon (underlined and bold), and the positions corresponding to extension inhibition products (underlined nucleotides).

the 80 S ribosome interacting with more mRNA 3‘ to the initiation codon than the 40 S ribosomal subunit was ruled out in a reconstituted system, where 80 S ribosomes yield an extension pattern identical to that obtained with 40 S ribosomal subunits when no amino acids or elongation factors are present (no elongation capability). One particular point to

note is that the stop site at position 15 is one that occurs during an extension reaction performed in the absence of ribosomes (see Figs. 2b, 36, and 5b) , possibly indicating a secondary structural element at this location.

Similarly, the radioactivity profile of a sucrose gradient used to analyze a ribosome-mRNA (P-globin mRNA) complex formed in GMP-PNP (1 mM) supplemented lysate is represented in Fig. 3a. Again, the formation of the ribosome-mRNA complex is cap-dependent, as addition of m7GTP inhibits the

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FIG. 3. a, sucrose gradient profile of ”P-labeled oligodeoxynucleotide-mRNA incubated with GMP-PNP-treated rabbit reticulocyte lysate. A ribosome-mRNA (@-globin mRNA) complex was formed in GMP-PNP-supplemented (1 mM) rabbit reticulocyte lysate over an incubation for 10 min at 30 “C in the presence or absence of 0.2 mM m’GTP, followed by sucrose gradient centrifugation. A graph of radioactivity uersus sucrose gradient fraction number is shown. The position of sedimentation for 80 S ribosomes, 40 S ribosomal subunits, and free 9 S globin RNA are marked at the top of the figure. b, extension inhibition analysis of the 40 S-mRNA complex obtained with GMP-PNP-supplemented rabbit reticulocyte lysate. Aliquots of peak tubes of radioactivity in the 40 S gradient region were analyzed as described under “Materials and Methods.” The nucleotide on the mRNA that corresponds to the migration position of the extension inhibition product and its position 3’ to the A in the initiation codon is represented on the right. A representation of the sequence of p- globin mRNA, below, indicates that initiation (underlined and bold) as well as the positions corresponding to the extension inhibition products (underlined nucleotides).


formation of this complex. However, in contrast to the anisomycin-dependent ribosome-mRNA complex, this complex migrates at roughly 40 S, indicating that this is the expected GMP-PNP-dependent 40 S-mRNA complex. Again, as previously stated, in the absence of inhibitor an incubation of mRNA in lysate for 10 min at 30 "C yields no detectable ribosome-mRNA complexes.

The GMP-PNP-dependent 40 S-mRNA extension inhibition pattern is represented in Fig. 3b. Extension stop sites are seen at positions corresponding to bases 15, 16, and 17 3' to the A in the initiation codon. This is consistent with the pattern obtained with the 40 S-mRNA complex region of the sucrose gradient when anisomycin was used.

When a-globin mRNA was analyzed by the same protocol, similar extension products were observed (data not shown). The extension inhibition pattern for 80 S-mRNA complexes accumulated in an anisomycin-dependent fashion yielded stop sites at 19 and 25-29 bases 3' to the A in the initiation codon. The 40 S-mRNA complexes yielded extension stop sites at 15, 16, and 17 bases 3' to the A in the initiation codon. However, the stop site at position 15 was not as strong as those observed at positions 16 and 17. Also, a stop site at position 18 was noted for the 40 S-mRNA complex. This may indicate that stop sites at positions 16 and 17 are more accurate reporters of the ribosome 3' border. Since a similar set of extension products obtained by the same technique for two different mRNAs could be obtained, it is viewed that this technique can likely be extended for analyzing other mRNAs as well.

A time dependence does exist for the formation of 40 S- mRNA complexes and 80 S-mRNA complexes. As seen in Fig. 4, a time curve of 0.5, 2, and 5 min at 30 "C in anisomycin- treated lysate yields a shift from 40 S- to 80 S-mRNA complexes, indicating that 80 S-mRNA complexes are formed via 40 S-mRNA intermediates as expected. The time course of 40 S-mRNA versus 80 S-mRNA complex formation indicates that the 40 S-mRNA complex forms much faster than the subunit joining step occurs. This could also be interpreted to be a ribosome scanning dependent time factor, but the exten-

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FIG. 4. Time curve: sucrose gradient profile of 32P-labeled oligodeoxynucleotide-mRNA incubated with anisomycin- treated rabbit reticulocyte lysate. Ribosome-mRNA (@-globin mRNA) complexes were formed by incubating the oligodeoxynucleotide-mRNA hybrid in anisomycin-treated rabbit reticulocyte lysate for 0.5, 2, or 5 min at 30 "C. After incubation the reaction mixtures were cooled to 4 "C on ice, and subjected to sucrose gradient centrifugation. Represented in this figure is a graph of radioactivity uersus the sucrose gradient fraction number.

sion inhibition products for all 40 S-mRNA and 80 S-mRNA complexes at each time point were identical (ribosome positioned at the initiation codon), indicating that the ribosome binding and scanning steps occur quickly relative to the 60 S subunit joining step.

A time curve was also performed for the GMP-PNP supplemented as well as nonsupplemented lysate (data not shown). Maximal GMP-PNP-dependent 40 S-mRNA complex formation is achieved by 2 min incubation at 30 "C. Further incubation begins to result in a slow progressive loss of detected 40 S-mRNA complex, possibly due to an oligodeoxynucleotide-mRNA unwinding activity. This would be consistent with the known RNA-DNA unwinding properties of eIF-4A, eIF-4B, and eIF-4F (23). As stated above, this loss of detected complex is not likely due to a problem with an RNase H activity or a phosphatase activity. An additional possibility here is that the GMP-PNP-dependent 40 S-mRNA complex can eventually surpass the GTP-dependent subunit joining step block and form an 80 S-mRNA complex, which begins to elongate and unwind the oligodeoxynucleotide-mRNA hybrid. However, a time curve of incubation with both anisomycin and GMP-PNP present yields only 40 S-mRNA complexes at all time points, indicating that the inhibition block conferred by GMP-PNP does not reverse to allow 80 S formation over the time course of incubations. Additionally, in the presence of GMP-PNP 40 S ribosomal subunits may bypass the initiation codon and proceed to unwind the RNA- DNA duplex.

Formation of a small amount of 40 S-mRNA and 80 S- mRNA complex (roughly 10% of that observed when translation inhibitors are utilized) can be observed when no inhibitor is added to the lysate (data not shown). However, these complexes are only detectable over the narrow time range of 0.5-2 min incubation at 30 "C. As stated previously, an incubation of 10 min at 30 "C results in no detectable complex. This observation can most easily be explained by a short time frame during which most ribosomes are initiating and are not yet elongating, allowing the oligodeoxynucleotide-mRNA hybrid to remain intact. The corresponding extension inhibition patterns obtained with these complexes are identical to those obtained with 40 S-mRNA complexes formed in a GMP-PNP dependent manner. This data indicates that the complexes observed, when using these translation inhibitors, reflect those intermediates that transiently exist normally in cell lysates active in protein synthesis.

Initiation Arrested Complexes (Initiation Site Recognition Deficient)-It has been previously shown that the antibiotic edeine is capable of facilitating the accumulation of 40 S- mRNA complexes in wheat germ lysate (6). In the same study it was indicated that the 40 S ribosomal subunit was not localized at any particular position on the mRNA. Edeine was added to nuclease-treated rabbit reticulocyte lysate, in this study, before mRNA addition. A short preincubation of lysate with edeine for 2 min at 30 "C was followed by an incubation in the presence of the mRNA-oligodeoxynucleotide hybrid for 2 min at 30 "C in the presence or absence of 0.2 mM m7GTP. A sucrose gradient profile of radioactivity of the above described experiment is represented in Fig. 5a. As seen in this figure a 40 S-mRNA complex is formed under these conditions. Furthermore, the formation of this complex is cap- dependent, as cap analog addition is capable of inhibiting complex formation.

When this 40 S-mRNA complex is analyzed by the extension inhibition assay (Fig. 5b) it is apparent that no limited site of interaction exists between the 40 S ribosomal subunit and the mRNA. Specifically, the 40 S ribosomal subunit does


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FIG. 5. a, sucrose gradient profile of 32P-labeled oligodeoxynucleotide-mRNA incubated with rabbit reticulocyte lysate treated with edeine. A ribosome-mRNA @-globin mRNA) complex was formed in edeine-treated (2 p ~ ) rabbit reticulocyte lysate over an incubation of 2 min at 30 "C. A graph of radioactivity uersu sucrose gradient fraction number is presented here for a complex formed in the presence or absence of 0.2 mM m'GTP. b, extension inhibition analysis of 40 S-mRNA complexes formed in edeine-treated rabbit reticulocyte lysate. An aliquot from the 40 S sucrose gradient radioactive peak region (identical to that seen in a) was analyzed by primer extension as described under "Materials and Methods." Extension reaction products are shown next to a dideoxynucleotide sequence ladder, which was taken from a longer exposure of the same gel. The nucleotide on the mRNA that corresponds to the stop site of the extension reaction, and its position relative to the A in the initiation codon is represented on the right. Below is a representation of the complete 5"untranslated region and a portion of the coding region for &globin mRNA. The position of the initiation codon (underlined and bold), and the positions corresponding to extension inhibition products (underlined) are indicated.

not strongly interact with the initiation codon. What seems to be the case, however, is that the 40 S ribosomal subunit can interact with the mRNA in a region 5' to the initiation codon. The strongest localization of 40 S subunits is seen by a set of 3 consecutive nucleotides 9,10, and 11 bases 5' to the A in the initiation codon. Alternatively, these stop sites can

be represented as 43,44, and 45 bases 3' to the cap structure. A number of other weak stop sites are located a t positions 19, 35, 48, and 54 bases 3' to the cap structure. Finally, a significant proportion of the 40 S-mRNA complex isolated by sucrose gradients centrifugation is seen as a full-length extension product upon extension analysis. The fact that a significant proportion of extension products are full length suggests that this population of 40 S-mRNA complexes isolated by sucrose gradient are not able to be analyzed by primer extension. This is most likely due to an interaction between the 40 S subunit and the mRNA that is stable enough to allow isolation by sucrose gradients, but not stable enough to with- stand the conditions of the primer extension reaction. Al- though the location of 40 S subunits on the mRNA is uncer- tain for this population, the possibility that the 40 S subunit is located 3' to the oligodeoxynucleotide is unlikely, because these complexes were formed in a cap dependent fashion. For the latter to be true, the 40 S subunits would have to interact with the cap structure of the mRNA and then proceed to jump past the RNA-DNA hybrid, which although possible, is unlikely. Instead, it is highly likely that the antibiotic edeine permits the accumulation of 40 S-mRNA complexes in a cap- dependent fashion, which are unable to localize stably at the initiation codon.

These observations are consistent with the notion that edeine interacts with the same position on the 40 S ribosomal subunit as does the initiator tRNA molecule (24-26). In so doing, it is likely that the absence of the initiator tRNA anticodon at the proper location on the ribosome prohibits the recognition of the initiation codon. It is believed that this codon-anticodon interaction is key in localizing the 40 S ribosomal subunit at the initiation codon (9). The ability to isolate 40 S-mRNA complexes, where the 40 S subunit is 5' to the initiation codon, in an edeine dependent fashion may be fully explained by a block in the ability to recognize the initiation codon, resulting in a situation where 40 S subunits are capable of mRNA binding, but not capable of localizing at the proper initiation site. These results are consistent with the ribosome scanning model that was originally proposed and supported by Kozak (7,8).

All results regarding the analysis of ribosome-mRNA initiation complexes indicate that the technique of primer extension inhibition is adaptable and quite sensitive for the study of the eukaryotic ribosome-mRNA interaction. Isolation of a complex from sucrose gradients before primer extension analysis allows the individual characterization of 40 S-mRNA and 80 S-mRNA complexes that are formed in crude rabbit reticulocyte lysate, a system which is highly efficient in translation.

Comparison of the stop sites from extension inhibition analysis between the published prokaryotic results and those demonstrated here for eukaryotic ribosomes yield similar results. Again, the extension stop site observed with a prokaryotic 30 S or 70 S ribosome is located 15 nucleotides 3' to the A in the initiation codon (1). The prokaryotic ribosome is thought to directly interact with nucleotides -20 to +13. Eukaryotic ribosomes are thought to interact with 30-35 nucleotides, as determined by nuclease digestion (27,28). The observation, here, of stop sites at positions 15, 16, and 17 indicates that the eukaryotic ribosome occupies a similar amount of space 3' to the initiation codon.

The occurrence of three stop sites as opposed to one may be due to a number of interesting possibilities. Three different stop sites could imply that the eukaryotic ribosome is not as strongly fixed to a particular location on the mRNA as is the prokaryotic ribosome. Another possibility would be that this


observation could be explained by a less rigid 3' border of the eukaryotic ribosome, resulting in a less abrupt collision event (over the span of 3 nucleotides). Finally, there could be different conformational states that the eukaryotic ribosome can assume. Data consistent with this possibility suggests that the ribosomal RNA structure changes upon ligand binding (24). While these possibilities are interesting, they are not easily discerned, and the actual reason that three separate stop sites occur matters little when interpreting the general location of the ribosome.

Regarding the time dependence of initiation complex formation a number of observations have been made. As previously stated, 40 S- and 80 S-mRNA complexes located at the initiation codon both transiently exist in a translationally active lysate. Evidence such as this would indicate that the steps of translation initiation subsequent to these complexes are rate-limiting. Those steps would be subunit joining, and some step after subunit joining but before elongation. Other data obtained in this study consistent with subunit joining being rate-limiting is the time course with anisomycin (Fig. 4). 40 S-mRNA complex formation is rapid in comparison to 80 S-mRNA complex formation. Furthermore, since all complexes can be localized at the initiation codon, scanning of the 40 S subunit from the 5' end of the mRNA to the initiation codon plays no time factor in this assay. At the same time, it is possible that, under normal conditions, a complex of the 40 S ribosomal subunit with the mRNA 5' to the initiation codon is not stable enough to be isolated by sucrose gradient centrifugation. Regardless, the isolation, in a noninhibitor dependent fashion, of 40 S-mRNA and 80 S-mRNA complexes that are localized at the initiation codon indicates that subunit joining and some step after subunit joining but prior to elongation are rate-limiting.

Other recent observations have indicated that there may be a critical initiation step subsequent to subunit joining, but prior to elongation. Analysis of nuclease-protected mRNA fragments in translationally active wheat germ lysate has revealed that a ribosome-mRNA complex at the initiation codon exists over a time period long enough to analyze by nuclease protection (29). However, while the identity of the ribosome was thought to be an 80 S ribosome in that study, the possibility that it was a 40 S ribosomal subunit cannot be ruled out. Analysis of this particular ribosome population by sucrose gradient would have clarified this point. Additionally, recent studies by Dr. Jack Hensold have involved the analysis of various ribosome-mRNA complexes from different cell lines under varying growth conditions.* Data from these experiments have also indicated that a population of 80 S ribosomes complexed to mRNA exists over a significant time frame. Under growth-repressed conditions, where translation is inhibited, this 80 S-mRNA complex is enhanced. It is not yet clear in those experiments, however, where this 80 S ribosome is located on the mRNA. While there has been little evidence accumulated to date for such a step, one known initiation factor may be involved at this site. Model assays have indicated that eIF-4D may be involved in formation of the first peptide bond (30, 31).

ATP-dependent Ribosome-mRNA Complex Formation-In order to assess the ATP dependence of the RNA interacting initiation factors (eIF-4A, -4B, and -4F) and the initiation events involving the 40 S-mRNA complex, an ATP-depleted reticulocyte lysate was used in conjunction with the primer extension inhibition assay. Hexokinase and glucose were used to achieve the ATP-depleted state. A preincubation of the ribosome-mRNA complex forming reaction mixture was car-

J. Hensold, personal communication.

ried out for 3 min at 30 "C in the presence of hexokinase and glucose. Subsequently, the oligodeoxynucleotide-mRNA hybrid was added to the reaction mixture and an incubation for 2 min at 30 "C under a number of varying parameters was carried out to form the ribosome-mRNA complex.

Fig. 6 represents an experiment that controls for the ATP status of the ribosome-mRNA complex forming assay. In this experiment a preincubation of the reaction with varying amounts of glucose is performed. Exogenous ATP-Mg2+ was also added back to the system in order to attempt to regain the initial ribosome-mRNA complex forming potential. GMP- P N P was also added to reaction mixtures after the ATP depletion step for two reasons. The first is that GTP may also be depleted under the conditions used in the ATP depletion step. Second, addition of GMP-PNP to the system allows for the accumulation of 40 S-mRNA complexes that can be isolated, quantitated, and analyzed by primer extension inhibition following sucrose gradient centrifugation when active 40 S-mRNA complex formation is capable of occurring. As can be seen in Fig. 6, GMP-PNP supplemented lysate allows for the accumulation of a significant amount of 40 S-mRNA complexes (represented as 100). Depletion of ATP from the system requires both hexokinase and 2 mM glucose, resulting in a substantial diminution of the 40 S-mRNA complex forming potential. This complex forming potential is partially regained when ATP"$+ is added back to the system subsequent to the ATP depletion step. The presence of lexose in molar excess over glucose is required to regain the ribosome- mRNA complex forming potential in this system when ATP- M$+ is exogenously added. The reason that the complex forming potential is not fully regained upon ATP addition may, in part, be due to a lack of aminoacylated initiator tRNA (Met-tRNAi). The complex forming incubation of 2 min may not be long enough to form this ATP-dependent aminoacyl-

FIG. 6. ATP depletion of lysate. The ribosome-mRNA complex forming mixture was depleted with respect to ATP as described under "Materials and Methods." Preincubation of the lysate, when performed, was done in the presence of 40 yg of hexokinase and varying concentrations of glucose, as indicated. GMP-PNP (1 mM) was then added to the reaction along with the [32P]oligodeoxynucleotide- mRNA (@-globin) hybrid. In some cases, after the depletion step (performed with hexokinase and 3 mM glucose), A T P - M e was added back to the reaction, along with 20 mM lexose. Complexes were formed during an incubation for 2 min at 30 "C and then subjected to sucrose gradient centrifugation as described in the legend to Fig. 2a. A graph of radioactivity versus sucrose gradient fraction number was then plotted. The radioactivity associated with 40 S ribosomal subunits was quantitated and is represented as a value relative to that obtained with GMP-PNP-supplemented lysate (+GDPNP lane: value of 100).


tRNA bond. From this experiment the most probable conclu- sion is that the variable in the system that is affected is the ATP concentration, and the ribosome-mRNA complex formation is dependent upon the presence of ATP. Additionally, primer extension inhibition analysis of the resulting 40 S- mRNA complexes indicates that, when a complex is present, the 40 S ribosomal subunit is located at the initiation codon (data not shown). From the studies with edeine, it is likely that a 40 S-mRNA complex, where the 40 S ribosomal subunit is not localized at the initiation codon, would be detected by this assay, if it were present. Because of this reasoning, it seems unlikely that the probable events of ribosome binding and scanning can be separated simply by partially depleting the system of ATP.

Efforts were then directed towards investigating the ATP requirement for the activity of the RNA interacting initiation factors, eIF-4A, -4B, and -4F. Fig. 7 represents an experiment that monitors the ribosome-mRNA complex as a function of an initiation factor-supplemented system in the presence or absence of ATP. As can be seen in this figure, in the absence of ATP no combination of initiation factor added enhanced the amount of ribosome-mRNA complex, but when ATP was added back to the system the combination of eIF-4A and eIF- 4B or the combination of all three factors significantly enhanced the level of ribosome-mRNA complex. Again, the location of the 40 S ribosomal subunit in all measurable complexes was at the initiation codon. The most simple interpretation of this data would indicate that the activity of eIF-4A, -4B, and -4F that leads to 40 S ribosomal subunits located at the initiation codon requires the presence of ATP either directly or indirectly. It should be noted that when factor additions were performed in the normal non-ATP- depleted lysate, a stimulation of ribosome-mRNA complex, only to the degree of 1.5-2-fold, was achieved upon addition

of eIF-4A and eIF-4B, or the combination of all three factors. An attempt was made to discern whether or not the activity

of eIF-4A, -4B, and -4F seen above directly or indirectly requires ATP, and possibly which factor possesses this ATP requirement. As seen in Fig. 8, addition of FSBA to the complex system (0.07 mM final concentration) did not affect the amount of factor (eIF-4A, -4B, and -4F)-mediated ribosome-mRNA complex formation. However, preincubation of the initiation factors with the same amount of FSBA (2 mM preincubation concentration) resulted in a 30-50% loss of the factor-mediated ribosome-mRNA complex formation capability. An activity loss of this magnitude is consistent with that seen due to the percent modification observed when preincubating the GTP utilizing enzymes phosphoenolpyruvate car- boxykinase, elongation factor la, or eIF-2 with the GTP analog FSBG (32). Also, while a preincubation of eIF-4B or eIF-4F alone with FSBA had no effect, preincubation of eIF- 4A with FSBA resulted in a significant loss of factor-mediated stimulation of complex formation. The possibility exists that neither eIF-4B nor eIF-4F reacts well with FSBA, and yet does require ATP for activity. The observation that pretreatment of eIF-4F results in no loss of factor-mediated stimulation may also be explained by an exchange of active eIF-4A for the FSBA-treated p46 subunit of eIF-4F, resulting in an active eIF-4F complex. It has previously been shown that, in the absence of the p46 subunit of eIF-4F, the p220 and p24 subunits are capable of interacting with eIF-4A, an observation consistent with the substitution of eIF-4A for the p46 subunit of eIF-4F (33). Also, it has been shown that a p46- deficient form of eIF-4F is capable of stimulating translation of globin mRNA in a reconstituted system with eIF-4A present (34). In any event, the most likely interpretation is that the ATP requirement of the eIF-4A, -4B, and -4F mediated

Y 150,

> FIG. 7. Initiation factor additions with an ATP-dependent

system. The ribosome-mRNA complex forming mixture was first depleted with respect to ATP by incubating the mixture 3 min at 30 "C with hexokinase and 3 mM glucose (-ATP and +2 mM ATP lanes). Then, initiation factor additions were made along with the addition of 1 mM GMP-PNP, [3ZP]oligodeoxynucleotide-mRNA (p- globin) hybrid, and the presence (+2 mM ATP lanes) or absence (-ATP lanes) of 2 mM ATP"< and 20 mM lexose. Complexes were formed during a 2-min incubation at 30 "C. Reactions were subjected to sucrose gradient centrifugation and radioactivity associated with 40 S ribosomal subunits was quantitated as described in the legend to Fig. 6. 40 S-associated radioactivity is represented as a value relative to that obtained in GMP-PNP-supplemented lysate (+GDPNP lane: value of 100).

n

P I- t e a

P - U

+ m

FIG. 8. FSBA-treated initiation factors. Lysate was first depleted with respect to ATP and subsequently l mM GMP-PNP, ["'PI oligodeoxynucleotide-mRNA (@-globin) hybrid, and, in most cases (+2 mM ATP lanes), 2 mM ATP"$+ and 20 mM lexose were added. At the same time eIF-4A, -4B, and -4F were added to the reactions with various components pretreated with 2 mM FSBA for 1 h at 30 "C as indicated. All factors were incubated 1 h at 30 "C whether or not they were treated with FSBA during this incubation. Complexes were formed during a 2-min incubation a t 30 "C and then subjected to sucrose gradient centrifugation. Radioactivity associated with 40 S ribosomal subunits was calculated as described in the legend to Fig. 6 and is represented here as a value relative to that obtained when ATP and all three factors were added to the system (+2 mM ATP, +eIF-4A/B/F lane: value of 100).


stimulation of complex formation is due to eIF-4A. Again, it should be noted that the location of the 40 S ribosomal subunit in all of these complexes is at the initiation codon.

Previously, an implication for eIF-4A as playing the ATP requiring role in initiation was derived from the observation that this initiation factor is sensitive to inactivation by the ATP analog, FSBA (35). Additionally, ATP has been shown to interact with eIF-4A by cross-linking studies (18, 36). Finally, eIF-4A displays an RNA-dependent ATPase and ATP-dependent RNA binding activity (13, 17, 37). Here, supporting evidence is given for eIF-4A as playing the primary ATP-dependent role in initiation steps resulting in a 40 S ribosomal subunit localized at the initiation codon.

Acknowledgments-We thank Dr. Rosemary Jagus and Mark Wil- son for supplying the antibiotic edeine, Dr. Terri Goss Kinzy, Dr. Jack Hensold, Dr. Hui-Kuo Shu, and Craig Cameron for helpful discussions, and Jane Yoder-Hill and Toni L. Bodnar for editorial assistance.

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