the control of globin synthesis in rabbit reticulocytes

PART IV. COORDINATED BIOSYNTHESIS OF rx AND p CHAINS

THE CONTROL OF GLOBIN SYNTHESIS IN RABBIT RETICULOCYTES *

Tim Hunt

Department of Biochemistry Tennis Court Road Cambridge, England

It is widely accepted that normal reticulocytes synthesize nearly equimolar amounts of the a- and /?-chains of globin,’ and that an adequate supply of hemin is provided by a controlled biosynthetic pathway starting with glycine and succinyl CoA.* It is also recognised that hemin synthesis is controlled by negative feedback on ALA synthetase,3 and that in the absence of hemin biosynthesis (as for instance during lead poisoning or iron deficiency) globin synthesis is severely ~ u r t a i l e d . ~ ~ In contrast to the coordination between heme and globin synthesis, little or no convincing evidence for the coordinated synthesis of a and chains seems to exist. Rather, the recognition that exces- sive synthesis of either chain without its partner occurs in the thalassemia

and the elegant experiments of Honig * argue strongly against such ideas as were once current: that a chains were necessary for release of /? chains from polysomes or vice versa.9, l o Similar ideas about how heme controls globin synthesis have also proven unlikely, and our attention today is almost wholly focused on the problem of the mechanism and control of the initiation of protein synthesis since all the evidence points to this step as being subject to control.

The initiation of protein synthesis occurs by a number of steps that seem to occur in a particular order. Several “initiation factors” catalyze these steps, although it is not always clear what the roles of the factors are. We have recently obtained evidence” that the steps occur as follows:

1) Met-tRNA, + GTP + 40s subunits P 40S/met-tRNAf/GTP 2) 40S/ met-tRNAJGTP + mRNA + 40S/mRNA/met-tRNA,/GTP 3) 40S/mRNA/met-tRNAf + 60s + 80S/mRNA/met-tRNA,

The evidence indicating that this series of reactions is followed is as follows: reticulocyte lysates, which make globin at essentially the same rate as intact reticulocytes, use met-tRNAf to initiate both the a and /? chains.25 The initiation of globin synthesis involves ribosomal subunits attaching to the end of the messenger RNA, and not the attachment of monomeric ribosomes.26 In lysates actively synthesising globin, a considerable fraction of the native 40s subunits carry met-tRNA,, and this met-tRNA, is used in preference to free met-tRNA, in initiation. Furthermore, under conditions of lack of added hemin, or in the presence of dsRNA, the met-tRNA, fails to associate with the 40s subunits; concomitant with this failure protein synthesis stops. However, in contrast to previous ideas, the 40S/met-tRNAf complex does not contain any mRNA as measured either by its sedimentation on sucrose gradients or its template

* The work was supported by Clare College, Cambridge and the Medical Research Council. I am a Beit Memorial Fellow.

223

224 Annals New York Academy of Sciences

activity. What is more, addition of mRNA to these complexes in the presence of 60s subunits and a full complement of “factors” converts them to 80s initiation complexes; that is, 40S/met-tRNAf complexes cannot exist as such in the lysate if free mRNA is also present, or even fragments of mRNA, since even the trinucleotide ApUpG can convert them to 80s complexes.

Knowing that initiation proceeds by an ordered series of steps allows the confident prediction that certain inhibitors will lead to the appearance of certain intermediates in exactly the same way that a mutant bacterium will accumulate intermediates in a metabolic pathway. We have found agents that will block each step in the pathway with high efficiency and specificity. As I mentioned, dsRNA and lack of hemin block the attachment of met-tRNA, to the 40s subunit; l2, Aurinetricarboxylic acid and poly-I block the attachment of mRNA to the 40S/met-tRNAf complex; and Edeine blocks the attachment of the 60s subunit to the 40S/mRNA/met-tRNAf complex (T. Hunt, R. J . Jackson, & S. Legon, unpublished observations). Edeine has proved to be a very useful inhibitor because of its very high specificity; it works at concen- trations roughly equimolar with the ribosomes and has no detectable effect on processes other than initiation; furthermore, it acts without a detectable lag. This makes possible the experiments described below that measure the relative assembly times of the a and ,!I chain of globin.

One of the curious features of globin synthesis is that there are (on average) different numbers of ribosomes attached to the two different messages; a chains are made on smaller polysomes than B chains.’* This finding, which has been confirmed in humans as well as in rabbits la where we first discovered it, suggests that there may be significant differences in the synthesis of the two chains. I t necessarily means at least two things. First, there is no possibility that the messages for a and /I chains are polycistronic. Second, since equal numbers of each chain are produced, and there are equal numbers of ribosomes making each chain, there must be more functional mRNA for a chains present in reticulocytes than mRNA for jl chains. Measurements of the relative amount of mRNA for each chain that is not being translated also show an excess of a-chain mRNA,24 which probably arises as a result of what appears to be a purely stochastic process of initiation on mRNA, although this is a hypothesis that ought to be tested. If it is really true that the output of a chains is equal to the output of p chains, and that equal numbers of ribosomes are making each kind of chain, it also follows that the difference in the sizes of the polysomes must result from a different frequency of initiation on the two messages.

This simple logic was lost on us in 1967-68, when we made many laborious, and as we thought successful, demonstrations17 that the rate of assembly of a chains was faster than the rate of assembly of p chains; however, the experiments were flawed by the absence of a rather simple and extremely obvious control; this was pointed out and corrected by Lodish,ls and our approach was undermined when he showed that there were differences in the time of appearance of labeled amino acid in the C-terminal peptides of the a and /3 chains, although it took an identical length of time for each chain to be released from the ribosome after completion. I don’t know why this is, whether it is related to Allan Morris’l’J finding of completed a chains attached to tRNA, for ex- ample, or whether the idea that there are two pools of tRNA for tyrosine is correct. It seemed to me that a new approach was needed to settle the issue, and so I exploited our new-found knowledge of the antibiotic edeine to measure the assembly times of the chains of globin in yet another way.

Hunt: Control of Globin Synthesis 225

Plan of the Experiment

Since edeine is completely specific for initiation, it is possible to measure the time taken to make a chain of globin (or of any protein for that matter) by measuring the rate of loss of nascent peptides from the ribosomes after the addition of edeine. In order to measure the relative amounts of nascent a and B chains present, I labeled a reticulocyte lysate with [35S]methionine for

0 60 120 146 TIME

FIGURE 1. The expected time course of loss of radioactivity in the methionine peptides of the a and p chains of rabbit globin.

Edeine is added at time 0; no loss of radioactivity is seen until the ribosomes that just escaped the edeine block pass the methionine residues at positions 32 and 55 in the a and p chains respectively. From then on, there is a linear decline in the activity of the peptides, until the aforementioned ribosomes release their chains. This figure is plotted assuming that there are equal numbers of nascent a and p chains, and that the rates of assembly of the two chains are identical.

five minutes at 30°C and then added edeine; samples were removed from the incubation at frequent intervals, and the ribosomes harvested by centrifugation. The nascent chains were digested with trypsin, the peptides separated by electrophoresis and counted. In order to correct for egregious losses during the procedure, the experimental samples were mixed with an appropriate amount of lysate labeled with [3H]methionine, so that the final results are expressed as a ratio of 35S/3H. The results that ought to be obtained are shown in FIGURE 1. Immediately after the addition of the inhibitor there is a lag,


which represents the time it takes the last ribosomes to initiate before the block to reach the methionine codons. There follows a period of linear decline until a basal level is reached, after which no more loss occurs. The exact time for assembly of each chain is most accurately estimated by timing how long it takes the ribosomes to lose exactly half the nascent chains after the addition of edeine, rather than measure the length of the lag or the time the line hits the baseline, since these points are not so well defined. It is important to have the initial and final level of counts accurately determined.

Experimental Details

The experiment was made three times, with minor modifications each time. The basic methodology is described in detail e1sewhere.l1 Reticulocyte lysates were incubated in the presence of added salts, amino acids, creatine phosphate, hemin and isotopically labeled methionine at 30" C. After five minutes incubation with [36S]methionine at 100 pCi/ml, the incubation was made 2 X 10-6M in edeine and mixed vigorously. Samples for analysis were withdrawn both before and after the addition of edeine, and protein synthesis stopped by dilution with 10 volumes of icecold buffer (25mM KCl, lOmM NaCI, lOmM Tris-C1 pH 7.5, 1mM MgCl,, 0.5mM dithiothreitol-SMISH buffer). In the first experiment, the buffer also contained lO-3M cycloheximide; in the last experiment, samples were frozen in liquid nitrogen before dilution. At this point in the last experiment, the samples were each mixed with an equal volume of lysate that had been incubated for five minutes with [3H]methionine and that had not had any inhibitor added to it; previous experiments had [3H]methionine-labeled globin added after the ribosomes had been harvested (this globin was prepared by an hours incubation of a lysate with [SHImethionine, followed by acid- acetone precipitation of the globin). The ribosomes were prepared by layering the diluted samples over 3 ml 15-30% sucrose gradients in SMISH buffer, and spinning at 50,000 rpm for three hours at 2 ° C in the spinco SW50.1 rotor. The red layer was carefully removed by pipetting, and the pellet and walls of the tube rinsed gently with water. The walls were wiped with kleenex to remove traces of free hemoglobin. The ribosomes were allowed to resuspend overnight in 1 ml of water, and then digested with RNase (100 pg/ml) and trypsin (100 pg/ml) in lOOmM NH,HCO, pH 9.0 for two hours at 37" C. The samples were lyophilised twice and spotted on paper for electrophoresis for one hour at pH 3.5 and 3kV. The spots containing methionine were located by autoradiography, cut out, and eluted with lOmM HCl. The HCl was removed by lyophilization, and the residues taken up in 0.1 ml of water for counting in 3 ml of scintillant (to1uene:Triton XlOO 2:l; 5g/l PPO). The counter was set to exclude tritium counts from the upper channel, but the lower channel counted 15% of the counts from the upper channel, for which corrections were made.

Results

FIGURE 2 shows the autoradiogram obtained from the early time points in the third experiment. Two points are worth noting: the immediate appearance of a heavy spot corresponding to methionine after the addition of edeine, which


results from the accumulated 40S/mRNA/met-tRNAf complexes (I will show elsewhere that the label is exclusively in the form of met-tRNA,), and second, the rapid disappearance of a spot that probably corresponds to the a-chain initiation peptide containing methionine. The 8-initiation peptide is probably hidden under the spot corresponding to uTS, which also become abruptly less intense after edeine is added and before the orderly loss of radioactivity due to chain completion is apparent.

FIGURE 2 shows the data from this same experiment; the expected features are present, though the actual levels of the two peptides at the start and the finish are not exactly as might be expected. The actual values obtained for the assembly times of the two chains (TABLE 1 ) were rather consistent, however, not only with each other but also with values obtained from the type of experiment in which the steady-state level of labeling of the ribosomes is compared with the accumulation of released radioactivity.20 They indicate that the times of assembly of the two chains are practically identical. These experiments also confirm that edeine does not affect the rate of movement of ribosomes along mRNA.

TABLE 1 THE ASSEMBLY TIMES OF THE a AND p CHAINS *

~~~

Experiment Assembly times (seconds)

1 2 3

a 65 61 63

B 67 65 64

* These assembly times represent the time calculated to make and release a complete chain. They were derived by measuring the time it took for the radioactivity in either aT5 or pT5 to fall to half their initial values. Other details of the experiments are described in the text.

It thus appears that the difference in size between the polysomes making u and 8 chains must have its explanation in a difference in the frequency of initiation on the two messages. Direct confirmation of this idea is not too easy, especially as it is difficult to purify the two messages. There is, however, another approach, albeit an indirect one. This consists in determining the relative output of u and 8 chains under conditions of ribosome scarcity, when there is a relative excess of mRNA over ribosomes. Under such conditions the amount of a particular chain that is made should simply be the product of the concentration of the mRNA for that chain and its affinity for ribosomes. Fortunately it is possible to inhibit ribosome function in such a way as to reduce the number of active ribosomes without altering the capacity of the survivors to function normally. This is done by reducing the rate of formation of 40S/met-tRNAf complexes with dsRNA or lack of hemin; those complexes that do form are functional.

A student in our laboratory, Ms. Jean Turner, has made a series of such experiments. She found that there was an excess of 8 chains made when synthesis was inhibited in the above mentioned way (TABLE 2). She also


confirmed Lodish's result, that elongation inhibitors led to relatively enhanced a-chain synthesis. Thus, the fact that /3 mRNA competes for ribosomes on better than equal terms when there are very few ribosomes available, and most of the mRNA is vacant, strongly emphasises the higher affinity that /3 mRNA has over a mRNA.

I

5 6 7 MINS

FIGURE 2. Autoradiogram of the tryptic digest of methionine-labeled nascent chains before and after the addition of 2 X 10-"M edeine.

The experiment is described in the text. Tracks 1-3 are digests of the nascent peptides taken before the addition of edeine; Track 4 was taken 20 seconds after adding edeine, and the next three tracks follow at 10-second intervals. Notice the disappearance of band A, which is probably the a-chain methioninecontaining initiation peptide, and the sudden appearance of free methionine (hydrolysed off the tRNA under the conditions of tryptic digestion). Electrophoresis was for one hour at 3kV on a standard sheet of Whatman 3 MM paper.

Discussion

The results of the experiments reported here confirm and extend the results of Lodish 18, 18 and suggest that the initiation sites of the a and /3 mRNA are different. Furthermore, these differences are recognised by a component or components of the lysate. I do not think it is necessary to postulate the exist-


h p t i d e A

Alpha T5

Beta !P5

Xethioninc

Origin

1 2 3 4 5 6 7

FIGURE 3. Result of the third edeine translation-time experiment. The experiment is described in the text. The data here differ from that expected (see FIGURE 1) owing probably to two major factors. First, the a-chain peptide, but probably not the p-chain peptide is contaminated with the p chain's methioninecontaining initiation peptide. This leads to the total loss of counts from the a chain being about 20% higher than expected. Second, the fact that there is not a complete loss of counts from either chain suggests a certain level of contamination of the ribosomes pellet with completed chains. Although these considerations flaw the experiments, the con- sistency of the results of the three experiments, and the fact that the slopes of the declining portion of the lines are the same strongly support the hypothesis of equal assembly times for the two chains.

TABLE 2

OF INHIBITION OF PROTEIN SYNTHESIS* THE b L A T I V E SYNTHESIS OF a AND p CHAINS UNDER CONDITIONS

Condition Rate of

synthesis a/! ratio

Control No hemin + dsRNA +4.5 mM Mg"

100 6.5 4.4 9 .O

1.00 0.6 0.89 1.78

* These experiments were done by Jean Turner. She made standard incubations with [9]methionine in the presence or absence of various inhibitors. She then mixed in a standard amount of lysate labeled for two hours with ['Hlmethionine, prepared globin, digested it with trypsin, separated the tryptic peptides by electrophoresis, located the spots by autoradiography, and eluted them for counting the TI/% ratio. As well as taking a sample for processing like this, she also determined the rate of protein synthesis in the presence of the inhibitors by removing a series of samples at four-minute intervals and counting the TCA precipitable counts. High Mg++ acts pri- marily as an inhibitor of elongation in the reticulocyte lysate.


ence of distinct initiation factors in order to account for the differences in handling of the two messages, since a single factor with a different affinity for the two sites would give the observed results. The more or less equal output of each chain is apparently achieved by the presence of excess a-chain mRNA, which is less frequently translated than the mRNA for chains. Both genetic evidence, and the evidence of the amounts of hemoglobin made in human a chain heterozygotes suggest that there are two genes for a chains but only one for jl chains.21 It is not impossible that the excess a-chain mRNA is made on a pure gene-dosage basis, and that its lower affinity for ribosomes has been selected for in order to compensate for this to achieve equimolar synthesis of the two chains. Actually, the presence of a pool of a chains has been noted in both rabbits and humans1122 so that the mechanism is still not perfectly balanced. We must await direct estimates of the amount of mRNA for each chain at various stages of erythrocyte development to determine the validity of this hypothesis.

If it is true that a mechanism exists to balance the overproduction of one mRNA over another, it is very doubtful whether it is possible to rectify a temporary imbalance, whether caused by amino acid starvation 23 or by heritable disease (the thalassernias).6, There seems to be an absence of what is normally called control-the existence of a mechanism that can match the synthesis of particular proteins to changing circumstances.

Proponents of the idea 23 that distinct initiation factors are required to promote the synthesis of each globin chain (and other proteins too) should be able to show that a cell-free system that cannot synthesize globin can be made to synthesize the appropriate chain when mRNA and the corresponding factor are added. Alternatively, and even better, a reticulocyte lysate titrated with an antibody to one of the purified initiation factors should stop making the corresponding chain, while continuing the synthesis of the other. Such an experiment avoids the criticism that highly fractionated systems inevitably draw with respect to their possible lack of physiological relevance. Neither experiment has yet been attempted as far as I know.

Acknowledgments

I thank Richard Jackson for many suggestions, Jean Turner for her skillful experiments, and Anne Brayley and Jack Brittain for patient assistance.

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the control of globin synthesis in rabbit reticulocytes

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