An Analysis of the Involvement of Purine Ribonucleotides in Eukaryotic Protein Synthesis .
An Undergraduate Biochemistry Laboratory Experiment T. Glen Lawson
Bates College, Lewiston, M E 04240, (207) 786-6293 Undergraduate students of biochemistry rarely encounter teaching laboratory experiments in which they can study directly the chemical mechanisms involved in the synthesis of proteins by cellular messenger RNA (mRNA) translation systems. In addition, most undergraduate biochemistry courses provide only classroom discussions of the relatively simple protein synthesis machinery of bacterial cells. The more complicated eukaryotic translation systems are often not considered in depth, though much recent research has demonstrated that the expression of a large number of genes in eukaryotic cells is regulated by events that occur a t the level of translation ( 1 4 ) . In the experiment described here, students carry out the synthesis of Brome mosaic virus (BMV) proteins in a rahbit reticulocyte cell-free system. Specific biochemical events in this elegant and complex process are demonstrated by measuring the influence of added purine ribonucleoside 5'4riphosphates (NTP's) and NTP analogs on the rate of protein synthesis. In carrying out this experiment, students also gain practice in the use of cell-free translation systems, radioactive tracers, and specific enzyme inhibitors, and they become familiar with the use of liquid scintillation counting. Background of Protein Synthesis The Translation of mRNA's
Tht:translation of mRNA's in eukaryotic systems can he dividcd into the ohases of'initiation, elongation, and termination. The most complex of these is initiation, which is also the most highly regulated portion of the process. The details of these processes, as they are currently understood, were published in recent reviews (13).These reviews include useful and detailed diagrams that can be beneficial in describing the protein-synthesis scheme to students. The initiation of translation requires the participation of a number of protein eukaryotic initiation factors (eIF's). Formation of a 435 Preinitiation Complex The process begins with the formation of a 435 preinitiation complex that results from the binding of a methioninel transfer RNAleIF-2 complex to an activated 405 ribosome subunit. The binding of the mRNAto be translated to the 43s complex is mediated by the recognition of the l-methylguanosine 5'-triphosphate (m7GTP) 5' cap structure of the mRNA by eIF-4F. This initiation factor, along with others, also catalyzes the adenosine 5'-triphosphat4ATP)-dependent disruption of higher-order structures of the mRNAmolecules (7, 8).Other initiation factors, including guanosine 5'triphosphate(GTP)-requiringeIF-5 (91,facilitate the binding of the 60s rihosome subunit to the mRNM43S complex, which yields the 80s initiation complex.
Elongation Elongation of the protein now begins. This phase involves a t least four elongation factors (eEF's). Each amino acid addition requires, in sequence, t h e GTP-dependent eEF-la-catalyzed binding of an aminaacyl-tRNAto the A site of the ribosome the dissociation of the eEF-la the formation of the new peptide bond the eEF-2-mediatedtranslocation of the growing peptide chain lkrmination Termination occurs when a stop codon is positioned in the A site of the ribosome. This codon is recognized by a releasing factor (eRF), which promotes the cleavage of the completed protein from the last-utilized tRNA in the rihosome P site. I t is possible that the hydrolysis of a GTP molecule is required for-the dissociation of eRF from the rihosome (6). Focus on Key Steps One approach to helping undergraduates to understand the complicated biochemical mechanisms associated with eukaryotic protein synthesis is to focus on the key steps, most of which involve purine rihonucleotides (Table 1).The importance of ATP and GTP for protein synthesis in eukaryotic systems can readily be demonstrated in the hiochemistry teaching laboratory. Lysates of rabbit reticulocytes a n d extracts of wheat germ a r e commercially available, and both are used by researchers for studies involving the in vitro synthesis of proteins. Rabbit reticulocyte lysate, which is used in the experiment described helow, is prepared by suspending whole cells in a hypotonic buffer to disrupt the membranes. The Table 1. Major Known Purine Ribonucleoside 5'-Triohosohate-Reauirina S t e m
Step
NTP required
aminoacyl-TRNA synthesis
ATP
elF-2B-mediated elF-2 activation
GTP
elF-4F binding to mRNA unwinding of mRNA structure by elF-4F,4A, and 4 8
ATP, m7GTP (cap structure) ATP
elF-5-mediatedbinding of the 60s ribosome subunit
GTP
eEF-1p-mediated eEF-1a activation
GTP
eEF-2-mediated ribosome translocation
GTP
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lysate is treated with micrococcal nuclease to destroy endogenous globin mRNA, and the preparation is supplemented with calf thymus tRNA and a n energy-generating system consisting of phosphocreatine and phosphocreatine kinase (10). Capped mRNA's, which are added to this lysate, are usually efficiently translated, and the rate of protein synthesis can be monitored by measuring the amount of a radiolabeled amino acid incorporated into acid-insoluble material as a function of time. The addition of purine NTP's or their analoes to these reaction mixtures can influence the rate of synthesis, thus showing their importance in the translation mechanism. Table 2 shows the results of such an experiment, which was carried out using the experimental procedure described below. Stimulation and Inhibition The RNA of BMV, a plant virus, was used in these reactions. BMV RNA is actually a mixture of four, capped genomic RNA's isolated from virions. The addition of ATP to the translation reaction results in a concentration-dependent stimulation of protein synthesis, whereas the presence of the nonhydrolyzahle ATP analog 6-adenylylimidodiphosphate (AMP-PNP), produces a marked inhibition of protein synthesis. These data clearly demonstrate the need for ATP hydrolysis in the translation process. The involvement of GTP in protein synthesis is shown by the increased rate of labeled amino acid incor~orationwhen the nvtction mixturei were supplemcnted'with CTP. Finallv. 5'-triohowhate " , the additlon of 7-mt~thvleuanoiincs " . . (m7GTP) results in a concentration-dependent decrease in the rate of protein synthesis. The m7GTP behaves a s a cap-structure analog and competes with the RNAfor binding to eIF-4F. This experiment duplicates one of the approaches originally used to discern the function of cap structures in the initiationof translation (11, 12). I t is also possible to examine the influence of these ribonucleotides on protein synthesis by analyzing the translation reaction mixtures by polyacrylamide-gel electrophor e s i s a n d autoradiography. T h e figure shows a n autoradiogram obtained by analyzing equal volumes of translation reactionmixtures incubated for 1h in the Dresence of ["5Slmethionine. Bands representing the four BMV Droteins are visible. and i t can be seen that both ATP and TI' stimulated the synthcsis o l the proteins, whereas AMI'-PNP and m7GW behaved a s inhihitors.
Table 2. Effect of Purine Ribonucleoside Triphosphates on the Incorporation of t 3 ~ ] ~ e u c i into n e BMV Proteins during the Translation of BMV RNA in Reticulocyte Lysatea RNA
added
NTP added
tritium incorporated. percentage of control (after:30 mini1 h ) b
no none 010 yes none 1001100 yes 0.2 m~ ATP 1521108 yes 0.4 m~ ATP 1721116 87.6161.8 0.2 mM AMP-PNP yes 0.4 mM AMP-PNP 68.1139.6 yes yes 0.2 m~ GTP 1181109 0.4 mM GTP 1381111 yes yes 0.2 mM m7GTP 81.3i75.2 yes 0.4 mM m7GTP 84.7165.1 'These data were obtained by fallowing the experimental procedure described in the text. b ~ hexpected e typical range of tritium incorporation is about 800 to 1600 cpm for the minus RNAcontrai and 5000 to 20,000 cpm for the plus RNAsamples. me minus RNAcpm value is used as a background and is subtracted from all measured cpm values.
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Experimental Procedure Caution: Students should wear disposable latex or PVC gloves to protect themselves from radioactive contamination and to orotect the exoerimental materials from skin oils.
This experiment requires the use of a small amount of a low-energy P-emitting radioisotope. Because the use of radioactive tracers in biochemistry research laboratories is virtually ubiquitous, providing students with an introduction to their use and handling has great educational value. The institution must be licensed for the use of tritium. Students who carrv out the ex~erimentmust first com~lete the required institutional radiation-safety training promam. (At our institution. we have a DreDared trainine unit that is incorporated inti the biochemi&ry laboratoh before starting this experiment.) A small quantity of low-level radioactive waste will be produced, and federal and state guidelines for the storage and disposal of these materials should be followed. The generation of waste can be minimized by having students work in groups of two or three, which does not impair the learning experience.
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Journal of Chemical Education
AJtoraa ogram of tne e ectrophoret c anarys s of 1 5-A-a1 q~otsof ,n v tro translal on react ons contaln ng BMV RhAm me oresence of 0 4 mM ATP, AMP-PNP GTP or m7GTP. Translat on react ons Rere caroresent ried out for 1 h as described in the text with ~~~~lmethionine
. .
as the laoeed am no acd Tne react on m xlLres ana yzed are ane 1 RhA alone, ane 2. RluA p JS ATP, lane 3, R N A p LS AMP-PNP lane 4 . R N A p l ~ GTP s ano lane 5 , R N A p JS m7GTP Arrons on tne left point to the viral protein bands. The locations of the size-marker proteins and their molecular weights are also indicated. The experiment described in the text can be expanded to incorporate this type of analysis. Adetailed procedure can be obtained from the aulhor by reauest.
Equipment and Supplies Most ofthe equipment required for this experiment is commonly found a t institutions equipped for basic education and research in biochemistry. This equipment includes udlu3table plperrors e g.. Gilsnn Piperman1
.
a p on it ant-temperature wnfrr bath a liquid scintillation counter
a cryogenic storage device (-70 "Cfreezer or liquid nitrogen container)
The experiment is facilitated by the presence of a vacuumfiltration manifold for use with 2.4-cm filter discs (e.a.. .. . model IT25 sampling manifold from Millipore,. Suclease-treated reticulocstr ~ P r o m e a aincludes ~ . lvsate . a supplemental amino acid mixture (minus either leucine or methionine). Each kit provides sufficient materials for a t least 150 reactions, and the lysate remains active for a t l e a s t two v e a r s a t -70 "C, if freeze-thaw cycles a r e N is inexpmsi\~etl'nmt!g;i,. ATP, AMPnvoided. B ~ RNA PNP, GTP, and m7GTPare avnilnblc from S i p a or several other suppliers. [3Hlleucine can be obtainedin aqueous solution that is stable for several years a t 4 "C (Amersham). Disposable 0.5-mL and 1.5-mL microfuge tubes and pipet tips must be autoclaved. All solutions should be prepared . . i n deionized water and sterilized. %nslation Reaction Mixtures The basic experiment can be completed in 3 or 4 h, not including the time required for liquid scintillation counting. The protocol, which uses t h e NTP concentrations listed in Table 2, is as follows. Translation reaction mixtures are set u p in 10 0.5-mL microfuge tubes on ice from aliquots of stock solutions prepared for each group. The components, added in order, are
vacuum-filtered through 2.4-cm glass fiber filters (GFIAor GF/B, Whatman) wetted with cold 10% TCA. The filters are washed three times with a Pasteur pipet full of cold 5% TCA (alternatively, the TCA solution can be prepared in a squeeze bottle) and two times with 95% ethanol. The filters are then thoroughly dried a t room temperature or in a 60-70 "C oven and placed in scintillation vials. The filters should be covered with 5 mL of scintillation cocktail. Many cocktail preparations will suffice, and the most e n v i r o n k e n t a ~ lHound ~ selection is a preparation that does not contain toluene. Five to ten minutes of conutiug time per sample is usually sufficient to provide counts per minute with a 20 error of under 2%. I t may be useful to have the groups share their data with all members of the class. This will provide a greater pool of data for each student to examine, and meaningful statistical analyses can be carried out on each data point. Expanding the Basic Protocol This basic protocol can be expanded to allow students to participate in the actual experimental design. Rather than being provided with the detailed procedure, students can be given a general outline of the experimental techniques and a list of the materials available to them. They can then design, based upon their classroom knowledge of t h e translation process, their own detailed protocols for assays to determine which NTP's and NTP analoes stimulate and which inhibit protein synthesis. Expansion of the list of available ribonucleotides to include purine mono- and diphosphates or even pyrimidines will spark considerable though; and discussioi; h y the stud e n t ~a i thev plan t h e ~ rinve.iti#ation. m:Gl)l' and m7GM1' will b(: almost >isinhibitor\, :is m:C;TI? AI)i'and GDP may have slight inhibitory eff& due to equilibrium considerations. AMP. GMP., and nvrimidine ribonncleotides will have little effect.) The experimental designs might also include more-comprehensive analyses of translation rate vs. nucleotide conthe students can pose and answer quescentration. tions concerning the relative effectiveness with which ATP, AMP-PNP, GTP, and m7GTP act to stimulate or inhibit translation. (In general, nucleotide concentrations in excess of 2 mM will produce little or no additional effects, so the concentrations of the stock solutions can be adjusted accordingly.) One scenario is to assign lah'groups one or two ribonucleotides for such in-depth investigations. Following the design and execution of the experiments, the class members can share and compare all of their resulting data and derive consensus conclusions. I n doing this, the students will function like a large research team.
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A.
acid) (HEPES~KOH, 7.5 to &e final volumes of 8 pL 0 . 7 5 or 1.5 @Lof nucleotide solution (2.13 mM ATP, AMP-PNP, GTP, or m7GTP) 6 pL of master mix 0.5 pL of either HEPESKOH (minus RNA control) or 0.10-0.50 &fiL BMV RNA Master Mix The master mix should be prepared on ice immediately before use. I t contains 2 0 mM of HEPES-KOH to give a final volume of 70 pL 3.15 pL of 1.0 M potassium acetate, pH 7.5
1.98 pL of 20 mM magnesium acetate, pH 7.5 1.84 pL of amino acid mixture minus leucine 2-5 pCi 13Hlleucine 54.1 @Lof freshly thawed reticulocyte lysate Procedure
To minimize handling of the radioisotope by students, preparation of the master mix can be carried out by a lahoratory professional. Do not centrifuge the reaction mixtures. The reaction mixtures are incubated in a water bath a t 30 "C. Aliqnots of 3 FL are removed from each mixture a t 30 min and 1h, and each is immediately added to 60 pL of 0.5 mM NaOH i n 1.5-mL tubes. These NaOH solutions are incubated a t 30 "C for 20 min to hydrolyze amino acids from charged tRNA's and are then placed an ice. The incorporation of [3Hlleuciue into newly synthesized protein is measured by trichloracetic acid (TCA~precipitation. To do this, 0.6 mL of 10% (wlv) TCA is added to the NaOH solutions prepared above, and the mixtures are incubated on ice for a t least 10 min. These solutions are then
ere
Acknowledgment The development of this experiment was supported in part by NSF-ILI grant award USE-9050695. Literature Cited 1. Henhey, J. W.B A n n v . Re". Rimhem. 1991,60.717-755. 2. Rhoads. R. E. J. R i d Chrm 1993,268.30174020. 3. Thach, R. E. Cell 1992.68,lii-1RO. 4. Kozak. M. Annu. Re0 C d i B i d 1992.8. 197-225. 6. Melofora. 0.: Hentzc. M W. Bioosmvs 1993.15.85-89. 6. Moldaue. K A n n u . Re". B i d e m . 1986.54. 1109-1149. 7. Ray, B. K: Lawson. T G.: Abramson. R. D.: Mernek, W. C.; Thaeh. R. E. J Bid Chem. 1986,261,1146611470. 8. Lawson, T. G.; Lee, K A ; Maimone, M. M.; Ahramson, R. d.: Dever, T E.; Merrkk, W C.: Thach. R. E.Rioehemzsliy 1989.28.4729-4734, 9. Chakravalti, C.: Maitra, U.J Btd. Chem. 1993,268, 1052P10633. 10. Jackson. R. J.; H u n t . T. Methods in Emymol. 1985.96.60-74. 11. Wehe& L. A ; Hickey, E. D.; Baglioni, C . J B i d C l h m 1978.263. 178-183. 12. Adsms. B. L.: Mornan. M.: Muthuknshnan. S.:Hecht. S. M : Shatkin.A. J. J. B i d
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