THE JOURNAL cm BIOLOGICAL CHEMISTRY Vol. 247, No. 23, Issue of December 10, pp. 7822-7829, 1972

Printed in U.S.A.

Regulation of Hemoglobin Synthesis

PREFERENTIAL INHIBITION OF a! An’D fl GLOBIN SYNTHESIS*

(Received for publication, May 12, 1972)

HARVEY F. LODISH: AND DAVID G. NATHANS

From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

SUMMARY

A large number of synthetic polynucleotides and poly- deoxyribonucleotides inhibit globin synthesis in cell-free extracts of rabbit reticulocytes; these include poly(rU), poly(rI), poly(rA), poly(dTh poly(dC), poly(d1) and poly(rU, r1). We show that all inhibit by blocking initiation of new chains, and have no effect on the rate of completion of nascent chains. Without exception all polymers inhibit preferentially initiation of CY chain synthesis. One interest- ing possibility is that ribosomes normally are heterogenous in their ability to translate a! and /3 globin mRNA, and that polynucleotides preferentially inhibit “o(-specific” ribo- somes. Alternatively, polynucleotides might interact with ribosomes so as to alter their ability to recognize the initia- tion sequences on the two mRNAs.

When the concentration of magnesium ion used in retic- ulocyte cell-free synthesis is 5 mu rather than the optimal 1.5 mu, there is preferential inhibition of production of ,8 globin chains; the ratio of CY to /3 chains produced is 2:l. Raising the concentration of magnesium resembles in its effects addition of antibiotics which preferentially inhibit elongation of polypeptide chains and also preferentially inhibit fl chain synthesis. Both result in more ribosomes than normal on each mRNA. Under such conditions initiation of protein synthesis is no longer a rate-limiting step and the relative production of proteins is proportional only to the relative amounts of messenger RNA.

We also discuss some long range therapeutic implications of this work in treatment of thalassemia.

Over 90% of protein synthesized by rabbit reticulocytes is hemoglobin, and the cells are regulated so as to synthesize essen-

*This work was supported by Grants AI-08814, AM-15322, AM-15929, and AM-05581 from the United States National Insti- tutes of Health.

$ Recipient of Research Career Development Award l-K4-GM- 50,175-01 from the National Institutes of Health.

0 Recipient of Career Development Award AM-35361 from the National Institutes of Health. Permanent address, Division of Hematology of the Department of Medicine, Children’s Hospital Medical Cknter, and t.he Department of Pediatrics, Harvard Medical School. Boston. Massachusetts. This work was Per- formed while a ?isiting ficientist in the Department of Biology, Massachusetts Institute of Technology.

tially equal amounts of the component cx and /3 globin chains (l-3). Recent work indicates that this regulation is complex. Although the rates of elongation of the two polypeptide chains are the same, as are the rates of release of the two completed

globin chains from the ribosome (4), indirect measurements show that rabbit reticulocytes contain about 1.5 times as much mRNA for (Y chains as for p (4, 5). However, ribosomes initiate pro- tein synthesis on each a! mRNA only about 65% as frequently as on each /3 mRNA (5). Since the amount of protein produced over a large period of time is proportional to the product of the concentration of mRNA and the rate of initiation of protein syn- thesis, this means that there is equal synthesis of the two chains, but that p chains are synthesized on polyribosomes which contain 1.5 times as many ribosomes as those translating QI mRNA (5,6).

The experiments in this paper add further support to the notion that synthesis of the two globin chains is controlled at the level of initiation of protein synthesis. We show that a large number of synthetic polyribonucleotides and polydeoxyribonucleotides inhibit initiation of globin synthesis in vitro. Without exception, all polymers preferentially inhibit initiation of o( globin synthesis. One interesting possibility is that ribosomes normally are heterog- enous in their ability to translate the two mRNAs, and that synthetic polynucleotides inhibit preferentially the “a-specific ribosomes.” Another is that the polynucleotides affect the ribo- somes so as to make them heterogenous in their ability to recog- nize the (supposed) different initiation sequences on the two mRNAs. Possibly, as we discuss, this is related to the normal decreased ability of an a! mRNA, relative to a fl mRNA, to initi- ate protein synthesis (5).

We also show that when the concentration of magnesium ion used in cell-free protein synthesis is 5 mM rather than the optimal 1.5 msr, there is preferential inhibition of synthesis of /3 globin chains. Our experiments indicate that raising the concentra- tion of magnesium resembles in its effects the addition of anti- biotics which preferentially inhibit elongation of the growing peptide chains, relative to initiation of synthesis of new chains (5). Under such conditions, as we showed previously, initiation of protein synthesis is no longer a rate-limiting step, and the rela- tive synthesis of proteins is proportional only to the amounts of the messenger RNAs.

MATERIALS AND METHODS

Reagents-Xl polynucleotides were purchased from Xles Laboratories. Concentration of solutions were calculated from the absorbance at 260 nm using conversion factors supplied by

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the manufacturers. According to the manufacturer, all had a molecular weight, measured by gel filtration, greater than 100,000. Pactamycin was a generous gift from CIBA; t3%]- methionine (20,000 mCi per mmole) was purchased from Amer- sham-Searle; all other radiochemicals were purchased from the New England Nuclear Corp. and used at the following specific activities: [14C]lysine (450 mCi per mmole), [14C]leucine (400 mCi per mmole), [3H]methionine (3,000 mCi per mmole), [YJisoleu- tine (220 mCi per mmole), and [3H]valine (2,500 mCi per mmole). Sources of all other chemicals were described previously (5, 7, 8).

Cell-free Protein Synthesis-Preparation of reticulocytes and their lysates and conditions for cell-free protein synthesis have been detailed previously (5, 7, 8). Each extract was tested in detail for the optimum concentration of magnesium acetate (1.5 mM to 2 mM for all lysates used here) and for hemin (10 pg per ml for all lysates). Reactions generally contained 19 nonradio- active amino acids (lo-4 M each) and one radioactive amino acid: [Y9]methionine (80 PCi per ml), [14C]lysine (4 PC1 per ml), [‘“Cl- leucine (4 j&i per ml), or t3H]methionine (15 &i per ml). Incu- bation was at 25”; irrespective of the radioactive label, incorpo- ration was linear for at least 20 min.

,411 other procedures utilized here have been detailed previ- ously: counting radioactive protein produced in cell extracts (7) ; preparation of [3H]methionine or [3H]leucine-labeled globin (5) ; separation of globin chains by chromatography on carboxymeth- ylcellulose (5, 9) ; peptide mapping procedures for separating and quantitating the methionine-containing peptides (uT5 and PT5 (5) ; and sucrose gradient techniques (5, 8).

TABLE I Preferential inhibition of 01 chain synthesis by polyribonucleotides

Reactions (60 ~1) contained [3%]methionine and the indicated

amount of polynucleotides. Incubation was at 25”. After 5-, lo-, 15-, and 20-min incubation, 5.~1 samples were withdrawn to meas-

ure the rate of protein synthesis. Incorporation was linear for

at least 15 min in all samples; the incorporation, relative to the sample without polymer, was calculated from this initial rate. The sample without polymer incorporated 30,700 cpm of [“%I-

methionine per 5-pl aliquot after 20.min incubation; background incorporation from samples not incubated was 1,400 cpm. TO

t,he remaining 40 ~1 were added 300,000 cpm of [3H]methionine- labeled globin, 1 cc of water, and 25 pg of ribonuclease. After incubation at 37” for 10 min, the samples were used to prepare

globin by the acid-acetone procedure (5, 12). After digestion with trypsin, the tryptic peptides cuT5 and pT5 were resolved by

paper ionophoresis at pH 3.5 (5). The ratio of or:/3 globin syn- thesized is calculated as follows: 36S:aH in ruT5; 3%:3H in pT5, as

described in detail previously (5).

Polymer

None

(d-A) b-C) 6% rG)

b-C) NJ) (r-4 b-1)

(dC) W,rI)

Cd11 (dT) f2 RNA

- I Amount

nmoles $hosphorus/ml

70

70 50

250

50 50 70

50 50 50

50 500

y0 Control proteir Ratio of a$ globin synthesis synthesized

100 1.00 90 0.98

84 0.94

81 0.92 61 0.72

53 0.82

44 0.67 38 0.54

35 0.76

27 0.64 21 0.41 21 0.44

61 0.70

7523

RESULTS

Inhibition of Protein Synthesis by Polynucleotides-It has been known for some time that single-strand polyribonucleotides, such as poly(U) and poly(I), preferentially inhibit initiation of globin synthesis in cell-free extracts of rabbit reticulocytes (10). Mathews (11) showed recently that single-stranded RNA from encephalomyocarditis virus also inhibits globin initiation, and we showed that the same was the case for bacteriophage f2 RNA (8). Neither viral RNA was translated by this system.

We then examined a variety of synthetic nucleic acids on cell-

free protein synthesis in extracts of rabbit reticulocytes (Table I, Column 3). At low concentrations (50 to 70 nmoles of phos- phorus per ml) poly(rU), poly(r1) and poly(rA) inhibited globin synthesis only slightly, and poly(rC) was even less efficient. Poly(dA) did not inhibit protein synthesis even at concentrations five times higher than those used in Table I. The polydeoxy- nucleotides poly(dT), poly(dI), and poly(dC) were much more effective inhibitors than their ribose counterparts poly(rU), poly(rI), and poly(rC). A control experiment in Table II shows that inhibition by polyribonucleotides was abolished when the RNAs were first digested with NaOH. This demonstrates that inhibition is not due to some (alkali resistant) impurity in the preparations. Likewise, as expected, inhibition by poly(dT) and other polydeoxyribonucleotides (data not shown) was unaffected by alkali.

In exploring further the inhibition by polydeoxyribonucleo- tides, we first showed that only the single-stranded polymers in- hibit (Table III). Whereas either poly(d1) or poly(dC) strongly inhibits globin synthesis, an equimolar mixture of the two, form- ing poly(d1) .poly(dC), has no effect. Likewise, addition of poly(rC) to poly(dI), forming poly(rC) .poly(dI), abolishes the inhibition of poly(d1) ; and addition of poly(dA) to poly(dT), forming poly(dA) .poly(dT), abolishes inhibition by poly(dT).

Polynucleotides Inhibit Only Polypepticle Chain Initiation-

Two types of experiments showed that all polynucleotides which inhibit globin synthesis affect only the initiation of polypeptide

synthesis. First, as shown by Fig. 1, a IO-min incubation (at

25”) in the presence of either poly(dT) or poly(dC) results in loss of larger polyribosomes, and concomitant increase in single ribo-

TABLE II

Effect of alkali digestion on inhibition by polynucleotides

Polynucleotides at 1 mg per ml were incubated for 18 hours at 37” in 0.1 M NaOH, then neutralized with an equal amount of HCl

and 0.03 M N-2-hydroxyethylpiperazine-N’-2.ethanesulfonic acid, pH 7.0. The control polymers were incubated in water at 0” for 18 hours, then the solutions were made 0.1 M NaCl, 0.03 M X-2- hydroxyethylpiperazine-N’-2-ethanesulfonic acid, pH 7.0. Reac- tions (60 ~1) contained [lJC]lysine. Aliquots (10 ~1) of reaction were taken at 5-, lo-, 15-, 20-, and 30-min incubation. Shown are results for the IO-min samples; results for the others were not

significantly different.

Polymer

y. Control synthesis

With polymer With polymer treated with NaOH

40 nmoles ph4wphorus/m1

None

NJ) (rA) (rC)

(d’J3

100 (3150 cpm) 39 39

82 19

92 87 94

20

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somes. All other inhibitory polynucleotides tested (at 50 to 70 nmoles of phosphorus per ml) yielded similar results: poly(rU), poly(rA), poly(dI), poly(rI), poly(rU,rI) (data not shown). This effect did not occur if emetine, an inhibitor of chain elonga- tion (5, 13), was added at 10e4 M together with the polymers (data not shown), a result indicating that the loss of polysomes requires protein synthesis.

While the experiment in Fig. 1 only indicates that polymers inhibit polypeptide initiation to a greater extent than chain elongation, the data in Fig. 2 show that polynucleotides such as poly(dT) do not inhibit the rate of polypeptide chain elongation at all. The antibiotic pactamycin, at a concentration of 7 x

lo-’ ill, completely blocks initiation of in vitro globin synthesis but does not affect completion of nascent chains (7, 14, 15). Incorporation of an internal amino acid, such as [l%]lysine, was normal for 2 min at 25” and then ceased (Fig. 2B) ; polyribosomes were completely converted into monosomes and subunits during

TABLE III

Only single-stranded polynucleotides inhibit globin synthesis

Reactions contained [14C]lysine; aliquots of 10 ~1 were taken at 5-, lo-, 20-, 30-min incubation. Shown are the results from the lo-min sample, which were not significantly different from the other aliquots.

Experiment 1 (poly- mers at 50 nmoles of phosphorus per ml)

Experiment 2 (poly- mers at 30 nmoles of phosphorus per ml)

-

A26C

02c

0.10

Polymer ZJ Control Synthesis

None 100 (2230 cpm)

~01~ (dI) 33 MY (r0 98 poly (dC) 57 poly(dI) + poly(rC) 96 poly(dI) + poly (W 104 None 100 (3220 cpm)

Poly NJ) 60 poly (d’-Q 38 ~01~ (W 92 ~01~ W-J) + poly (da) 94 wG(dT) + POIY (d-4 88

Control Reaction

this period. Hence, the brief incorporation of [14C]lysine in the presence of pactamycin represents only completion of nascent chains (7, 15). Fig. 2B shows that poly(dI), poly(rI), or poly- (dT), when added together with pactamycin, had no effect on the rate of completion of nascent chains, as compared to the re- action which contained only pactamycin. Similar results were obtained with the other polynucleotides tested: poly(rU), poly- (rA), poly(dC), and poly(rU, r1). We conclude that all of the polynucleotides which inhibit globin synthesis affect only the initiation of polypeptide synthesis.

Inhibition by the polynucleotides was independent of the con- centration of magnesium ion (Table IV). This result shows that these compounds do not inhibit globin synthesis by chelating magnesium ion.

Polynucleotio?.es Preferentially Inhibit Synthesis of CY Globin-A rapid and sensitive peptide assay was used to measure relative synthesis of a and ,!3 chains. The method takes advantage of the fact that each rabbit globin chain contains only 1 methionine residue in an internal position (16, 17), and the two tryptic pep- tides containing methionineTT5 and PT5--can be resolved by paper ionophoresis at pH 3.5 (5). In vitro reactions labeled with [35S]methionine were mixed with [3H]methionine globin. From the ratio of 35S:3H in aT5 and flT5 the relative synthesis of a! and fi chains was calculated. Tables I and V show that all syn- thetic polynucleotides preferentially inhibit the synthesis of Q( globin chains. When several different nucleic acids were com- pared at about the same concentrations (Table I) or when the same polynucleotide was studied at several concentrations (Table V) the degree of inhibition of over-all globin synthesis correlated with a reduction in the ratio of cy to fi chains produced.

To confirm these results using another technique, protein syn- thesized in vitro in the presence of polynucleotides and labeled with [14C]leucine was mixed with a small amount of hemoglobin uniformly labeled with rH]leucine. Globin was isolated and the two chains separated by chromatography on carboxymethyl- cellulose. The ratio of 14C:3H in each globin peak is a measure of the relative amount of chain synthesized. Table VI shows that each of the four polynucleotides tested preferentially in- hibits synthesis of a globin chains.

Poly dT 40 nmoles P/ml

f Poly dC 70n moles P/ml

-.

+

Fraction

-

FIG. 1. Effect of polynucleotides on polysome size. Reactions After 10 min at 25’, 200 ~1 were taken to a tube containing 1.0 ml (240 ~1) contained [14C]lysine and polymers as described. Ali- of Buffer B (5, 8) and layered atop a 36-ml 15 to 50$& sucrose quots (10 pl) were removed after incubation of 5, 10, and 15 min. gradient in the same buffer. Centrifugation was at 4” for 534 The reaction with poly(dT) incorporated [l*C]lysine into protein hours in the Beckman SW 27 rotor. Optical density at 260 nm at a rate 35y0 of the control; that with poly(dC), 41oj, of control. was monitored continuously as described previously (8).

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No Druqs Pactamvcin 10m7M

1 ’ 1 I 4 8’ 12

- :‘J (No drug or polymer) 400 I

A

Minutes

FIG. 2. Effects of polynucleotides on rate of completion of lo+ M pactamycin. Polynucleotides were used at 70 nmoles of nascent chains. Reactions (0.12 ml) containing [l%]lysine were phosphorus per ml. Note the difference in scales between A and described previously (5, 8); 10 pl aliquots were counted. Reac- B; the reaction with no polymer or drug at all (No polymer, panel tions in the Zeft panel (A) contained only the polymers indicated, A) is replotted as an interruped line on the right scale (No drug whereas those on the right (B) (except the interrupted line labeled or polymer). O-O, no polymer; A-A, poly(dT); O-O, No drug or polymer) contained, in addition to any polymer, 7 X poly(r1) ; A--A, poly(d1).

TABLE IV TABLE V

Inhibition by polynucleotides is independent of magnesium Polynucleotides preferentially inhibit synthesis of a globin chains ion concentration Reaction conditions and measurement of the ratio of a:p chains

[X!]Lysine was the label. Aliquots (10 ~1) of reactions were synthesized were exactly as in Table I.

taken at 5-, lo-, 15-, 20-, 30.min incubation. All reactions were linear for at least 15 min; shown are the results for the IO-min aliquots. All polymers were used at 50 nmoles of phosphorus per

ml.

Polymer

Magnesium acetate (‘% control synthesis) None Polymer Poly (r1)

0.5 m* 1.5 rnY 3.3 rnM

None 100 (2780 cpm) 100 (2920 cpm) 100 (1660 cpm) Poly (dT) Poly (rU) 65 52 48 Poly (rA) 48 47 54 Poly (dT) 25 24 23

Poly(d1)

Bacteriophage f2 RNA Also Prejerentially Inhibits a Chain

Synthesis-Earlier work in this laboratory showed that f2 RNA inhibits initiation of globin chain synthesis (8). Table I demon-

-

Amount

n??wles phSjhWUS/WLl

30 70

103 10 20

30 50 10

20 30

50

70 Control synthesis

100 1.01 36 0.57 14 0.38 10 0.37

87 1.01 76 0.96 59 0.84 24 0.50 95 0.99 53 0.68 41 0.63 18 0.41

Ratio a:@ globin synthesized

strates that f2 RNA also preferentially inhibits synthesis of a globin chains. This result was confirmed by the experiment shown in Fig. 3; an in vitro reaction containing f2 RNA was labeled with [35S]methionine while a control reaction was labeled with [aH]methionine. The samples were mixed together, globin was isolated, and the (II and p chains separated by chromatog- raphy on a carboxymethylcellulose column. Addition of f2 RNA resulted in synthesis of considerably less QI chain, relative to @, than did a control reaction.

Effect of Magnesium Ion on Synthesis of LIP and p Globin-In

virtually all of our reticulocyte extracts the optimum concentra- tion of magnesium ion required for cell-free protein synthesis is

1.5 mrvr to 2.0 m&f (see Figs. 4 and 5). At 5 mM magnesium pro- tein synthesis proceeds linearly, at a rate which is 25 to 40yo that which obtains at the optimum concentration of magnesium.

Two different techniques were used to measure synthesis of a and /3 globin chains as a function of magnesium ion concentration. Both yielded the same result; at 5 mM Mg+f the ratio of a to ,6 chains synthesized was greater than 2 : 1, whereas at the optimum Mg++ concentration the ratio was about 1 :l. (a) The experi- ment in Fig. 4 utilizes the incorporation of [35S]methionine into tryptic peptides (rT5 and /3T5 (the technique used previously in Tables I and V) as a measure, respectively, of a! and fi chain synthesis. (b) Protein synthesized in vitro at different magne-

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TABLE VI ---- Column chromatography of globin chains synthesized in

presence of polynucleotides

Reactions (0.12 ml) containing [r*C]leucine were incubated at

25” for 20 min; aliquots of 10 ~1 were withdrawn after 5-, lo-, and 20-min incubation to measure the rate of protein synthesis. Incorporation in all samples was linear for at least 10 min and the

incorporation, relative to the sample without polymer, was calculated from this initial rate. An amount of reaction mixture which contained about 100,000 cpm of protein was added to about

300,000 cpm of [3H]leucine-labeled hemoglobin. Preparation of globin and separation of the two globin chains by chromatography on carboxymethylcellulose was as described in the legend to Fig.

5. At least four tubes from the peaks of c\! and p chains were counted. The ratio of cu:p globin chains produced is the ratio r4C:aH in or:14C:3H in 0 globin. I-LA

I 2 3 4 5

Mg++ [mM] POlylll~I

None

poly (d’J3 POSY (d1)

~01~ (dC)

POSY (r1)

Y

C3H)

x OS (

I

x

)-

j-

Amount 70 Control synthesis

Ratio a:8 globin synthesize

100 0.96 40 22 0.59

40 25 0.55 75 63 0.69 50 22 0.58

FIG. 3. Inhibition of 01 chain synthesis by f2 RNA. A 509~1 reaction containing [%]methionine and 25 rg of f2 RNA and a 700~~1 reaction containing rH]methionine were incubated at 25” for 15 min. The reactions were mixed, globin was isolated, and the chains were separated by chromatography on a column (1.0 X 30 cm) of carboxymethylcellulose using a pyridine-formate gradient elution as detailed previously (5). Fractions of 4 ml were collected; 0.5 ml wasmixed with 10ml of Aquasol and counted in a scintillation counter. X-X, 3H; O-O, %3.

sium concentrations and labeled with [14C]leucine was mixed with a small amount of hemoglobin uniformly labeled with [3H]- leucine. Globin was isolated and the two chains separated by chromatography on carboxymethylcellulose. The ratio of 1% :3H in each globin peak is a measure of the relative amount of chain synthesized. Fig. 5A shows that at 1.5 InM magnesium the ratio of ar :p globin synthesized was 1.03, whereas (Fig. 5B) at 5 mM the ratio is 2.12.

Mechanism of Ilthibition by High Magnesium Ion Concentra- tions-The above results indicated that high concentrations of magnesium ion appeared to resemble the effects of antibiotics-

FIG. 4. Effect of magnesium acetate concentration on synthesis of o( and p globin. Reactions (60 ~1) containing [%]methionine and the indicated concentration of magnesium acetate were incu- bated at 25” for 20 min. To each was added l,OOO,OOO cpm of [3H]methionine-labeled globin. The relative synthesis of a and p chains, measured by the ratio of 35S:3H in tryptic peptides orT5 and PT5, was, as described in the legend to Table I. O-O, orT5; o---o, pT5.

as cycloheximide and sparsomycin-which specifically inhibit elongation of peptide chains (5, W-21) ; all inhibit preferentially synthesis of ,8 globin chains (5). Hence, it was of interest to define more closely the mechanism of inhibition of globin syn- thesis by magnesium ion.

Fig. 6 (top panel) confirms previous results (22) that, after in- cubation at 1.5 InM magnesium, polysomes synthesizing globin are comprised of clusters of three to five ribosomes. By con- trast, in reactions containing 4 mM magnesium the polysomes are considerably larger than normal (predominantly five to seven ribosomes) and a larger fraction of the ribosomes are found on polyribosomes. Similar results (not shown) were obtained at 5 mM magnesium. Since the number of ribosomes per mRNA is proportional to the rate of polypeptide chain initiation and in- versely proportional to the rate of chain elongation and termina- tion, we conclude that the primary effect of the high magnesium concentration is to inhibit preferentially elongation of the grow- ing peptide chain. It is important to note that at 1.5 mM mag- nesium, fl chains are synthesized on polyribosomes significantly larger than those producing a chains (Fig. 6, bottom panel) ; this result was shown previously by Hunt et al. for intact reticulocytes (6) and by ourselves for cell-free protein synthesis (5). By con- trast, at 5 mM magnesium both chains are synthesized on the same classes of larger polyribosomes; note also that, as expected, there is twice as much nascent a: chain as 0. Hence, as we dis- cussed in detail below, high concentrations of magnesium ion re- semble in its effects on globin synthesis antibiotics which pre- dominantly block elongation of the growing peptide chains.

DISCUSSION

In this study we examined the effects of polynucleotides and of magnesium on cell-free synthesis of hemoglobin. In brief, our new findings are: (a) a large number of synthetic polyribo- and polydeoxyribonucleotides are specific inhibitors of initiation of globin synthesis; these include poly(dT), poly(dI), poly(dC),

poly NJ), poly b-1) , ~01~ W) , po]y (rA) , poly NJ, r-1)) and PO]Y- (rA,rG). (b) All of these polynucleotides specifically depress initiation of Q chains relative to /3 chains. Comparing one poly-

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IA

1’%) (‘HI 0 i x

$ IOOO! 10,000 \ 2 000 8,000 i

1 I I

CONTROL a/p=1.03

. . . ..*....0.098 +0.004

l ..*.* -0. 0.095

+o.ooz

50 60 70 FRACTION

FIG. 5. Synthesis of a! and p globin chains. A, at 1.5 mM mag- nesium acetate; B, at 5.0 mrvr magnesium acetate. Reactions containing [i4C]leucine were incubated for 10 min at 25”. The samples were chilled on ice and mixed with 5 mg of rabbit hemo- globin labeled with [zH]leucine (prepared by labeling reticulocytes from the same rabbit) ; globin was prepared by precipitation with acetone (12). Chromatography was on a column (1.0 X 30 cm)

1.5mM Mg ++ Reaction

0.1 -

40mM Mg++ Reaction

1 I 5 mM Mg++ REACTION

a//3=2.12 (‘4CV(3Hl

/ l

0. I 0

. *

. . . . . l 0.085 !

0.90

. kO.004 0.08 0.07 0.06 0.05

'.'"""'~0.040- 0.04

600

40 50 60 70 FRACTION

of carboxymethylcellulose (Whatman CF32) ; elution was per- formed as in References 5 and 9, except that all buffers contained 0.02 M 2-mercaptoethanol. Fractions of 4 ml were collected; 0.5 ml was mixed with 10 ml of Aquasol and counted in a scintillation counter. The average 14C:3H ratio is shown above each peak. 0~0,14C radioactivity; X-X, 3H radioactivity; 0, 14C:3H.

nucleotide at different concentrations (Table V) or several polynucleot’ides at the same concentration (Table I), there is a good correlation between inhibition of over-all globin initiation and reduction in the ratio of Q! : fl chains synthesized. In some cases there results a ratio of a to /3 chains of less than 0.4 to 1. (c) By contrast, high concentrations of magnesium ion in the in vitro reaction mixtures specifically depresses synthesis of /3 chains, resulting, at 5 mM, in a ratio of (I! :/3 produced of over 2 to 1. (d) In contrast to the polynucleotides, high concentrations of mag- nesium ion appear to inhibit elongation of nascent globin chains to a greater extent than initiation of new chains; polyribosomes are larger than in control reactions (containing 1.5 mM magne- sium). At the high magnesium concentration a and fl chains are synthesized on the same sized classes of large polyribosomes, whereas normally 0 are made on larger polysomes than (Y.

Certain facts concerning regulation of (Y and p chain synthesis in reticulocytes influence the interpretation of these findings. These have been briefly described in the introduction and in- clude the following.

1. Mammalian reticulocytes normally contain a slight excess of (Y globin chains, but they synthesize nearly equal numbers of a: and /3 chains (l-3).

2. Hemin-supplemented cell-free extracts of rabbit reticulo- cytes will synthesize globin for prolonged periods (23). At the optimum magnesium concentration for cell-free synthesis (1.5 to 2.0 mM) there is equal synthesis of the two globin chains (4, 5).

3. In intact rabbit reticulocytes, p chains are synthesized on polyribosomes containing about 1.4 times as many ribosomes than do those producing LY chains (6). This dissociation has been confirmed as well for cell-free synthesis of globin by crude reticulocyte extracts (at 1.5 mM magnesium ion) (5), for whole human reticulocyt’es (24), and for rabbit and human bone mar- r0w.l

4. Although earlier studies suggested differences in the rates of elongation of nascent c~ and fl chains (25), recent measure- ments indicate (4) that there is no difference in the rate of elonga-

1 H. F. Lodish and D G. Nathan, manuscript in preparation.

Fraction

FIG. 6. Distribution on polyribosomes of nascent 01 and fi chains. Left, 1.5 InM magnesium acetate; Right, 4.0 mM mag- nesium acetate. Two OX-ml react,ion mixtures containing [l”C]- isoleucine (5 pCi per ml), [sH]valine (50 pCi per ml), and 18 non- radioactive amino acids were incubated for 10 min at 25”. One c.ontained 1.5 mM magnesium acetate, the other 4.0 rnM. The reaction mixture was chilled rapidly in ice. One milliliter of Buffer A (0.025 M N-2-hydroxyethyl piperazine-N’-2-ethanesul- fonic acid, pH 7.0; 0.05 M KCl, 0.002 M magnesium acetate) was _ added and the solution was layered on a-3G-ml linear sucrose gradient (15% w/v to 30% w/v in Buffer A). Centrifugation was For 15 mm ai 26,500 rpm in’ the SW27 rotor of the Be&man L-3 ultracentrifuge; temperature was maintained at 4”. Analysis of the polysome fractions has been described in detail previously. From the ratio of 14C:3H in each fraction one can calculate the ratio of nascent OL to 6 chains. Plotted for each fraction is the total amount of [aH]valine radioactivity in nascent o( or nascent fl chains, divided by the average number of valine residues per nascent chain. Hence these are a measure of the number of moles of nascent chain. The assumptions and details of this type of calculation have been given in detail (5, 6).

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tion of the nascent o( and /3 globin chains, nor is t,here a difference in t,he rate of release of completed globin chains from polyribo- somes. Under these conditions each 6 n1RN.X contains 1.4 times more ribosomes than each cy mRNA. Hence, each fi mRN&i initiates synthesis in any time interval of 1.4 times as many globin chains as does each a! mRNX. Reticulocytes must contain about 40% more (Y mRNA than 0 mRNX in order for the cells to synthesize equal numbers of the two hemoglobin chains (4).

5. Previous indirect measurement of the relative amounts of functional (Y and /3 mRNA in the reticulocyte confirms the above conclusion that there is more functional (Y mRNA.

These experiments (5) utilized seven ant,ibiotics which, at inhibiting concentrations, all reduce the rate of propagation of nascent globin chains but did not directly inhibit initiation of new chains. This resulted in accumulation of large polyribo- somes, in which the mRNA molecules were sat,urat,ed with ribo- somes. Under these conditions protein synthesis was not limited by chain initiation and the relative amounts of t,he two globin chains produced should be proportional to the relative amount of OL and fl mRNA. Indeed all of these drugs induced a change in the ratio of cr:/3 globin synthesized from the normal 1.0 to about 1.4, and this ratio is believed to be the ratio of the amounts of the two mRNAs (4). In the presence of any of the seven trans- lation and both types of globin chains were made on the same classes of very large polyribosomes which contained relatively more nascent OL chains than p.

Effect of Magnesium Ion on Cell-free Globin Synthesis-There exists a large body of earlier work, much of it done with syn- thetic polynucleotides in bacterial extracts, which indicates that the initiation of protein synthesis and the elongat,ion of nascent chains may have different magnesium optima (reviewed in Reference 26).

To a first approximation, high concentrations of magnesium ion in reticulocyte extracts resemble the translation inhibitors mentioned above. Polysomes are larger than normal (Fig. 6), a result indicating that chain elongation or termination, or both, is inhibited, relative to chain initiation. Likewise, o( and fl chains are both synthesized on the same classes of large poly- ribosomes. The synthesis of more a chains than p could be explained, in part, by the same logic as used above for antibiotics. However, the highest ratio of CY:~ chains synthesized in the presence of any of the translation inhibitors was 1.60 (5), a value close to the ratio (1.5:1) of Q! to @ mRNX which was postulated from earlier studies (5). By contrast, at 5 mM magnesium the ratio of o( to p chains produced was greater than 2:1, a number which exceeds the (postulated) ratio of a! to p globin mRNA. One possibility is that high concentrations of ma.gnesium, while primarily slowing ribosome movement along the mRNA, also directly inhibit initiation of 0 chain synthesis. Some support for this explanation is in the lower right panel of Fig. 6 where it can be seen that at 4 lllM magnesium each a! mRNA contains, on the average, somewhat more ribosomes than does each /3 mRNA.

It is clear, however, that a detailed study of t,he effects of magnesium ion on each stage of protein synthesis is required in order to settle this question.

Synthetic Polynucleotides-Synthetic polynucleotides could in- hibit initiation of globin synthesis either by binding to (some region of) the mRNA, preventing its attachment t.o ribosomes, or interacting in some manner with ribosomes preventing or altering attachment to mRNA or polgpeptide chain initiation. The second possibility is strongly favored for the following rea-

sons. (a) A large number of synthetic and natural polynucleo- tides inhibit, rendering unlikely any specific interaction with the mRNAs. (b) Inhibition is preferential for a chain synthesis; were inhibition due to interaction with the mRNb, it is difficult to see how so many different polynucleot~ides could interact preferentially with a! mRNA. (c) Other polyanions, such as dextran sulfate and polyvinyl sulfate, are potent inhibitors of chain initiation in other systems.

One explanation for the fact that all polynucleotides prefer- entially block initiation of a! globin synthesis is that reticulocyte ribosomes are normally heterogenous in their function, some specific for translation of CY mRNA, some for @. Such heter- ogeneity could be caused by ribosomal proteins which might be present on only a fraction of all ribosomes (27). According to this notion, ribosomes which translate a! mRNA would be more sensitive to inhibition by polynucleotides than the “p ribosome.”

A second possibility is that ribosomes normally are functionally homogenous but that synthetic polynucleotides modify the ribosome in such a way as to modify its ability to recognize different initiation sequences on different mRNAs. Possibly the specific inhibition of (Y chain initiation is related to the fact, discussed above, that cx ntRNA is normally disfavored, relative to p mRNA, in polypeptide chain initiation and, presumably, in attachment to ribosomes. Normally, then, ribosomes can differentiate between the initiation sequences on o( and fl mRNA, possibly because of different primary sequences on either side of the supposed AUG initiation codon (7, 28, 29), and possibly because the secondary structure of the Q! mRNA is such as to restrict ribosome attachment to the initiator site (30). The synthetic polynucleotides would affect the ability of ribosomes to differentiate between these two initiation sites.

It was of interest that, in general, polydeoxynucleotides in- hibited globin initiation better than the corresponding polyribo- nucleotides. Possibly this is related to the recent findings that some single-stranded DNA molecules can bind to ribosomes and support some of the steps of polypeptide chain initiation, al- though they apparently cannot direct synthesis of long poly- peptides (31, 32).

Implications of This Work-Studies on the basis of the effects of various agents on (Y and fi chain synthesis may provide im- portant information concerning the physiology of protein syn- thesis. Such inquiries may also have long range therapeutic implications. Patients with thalassemia suffer from inherited underproduction of one of the globin chains and relative over- abundance of the other. This leads to a combination of hypo- chromia and hemolytic anemia secondary to formation of in- clusions in the reticulocytes; the latter is due to precipitation of the uncombined chains produced in relative excess (33). Agents which might selectively inhibit the synthesis of the chains pro- duced in excess would also reduce the extent of inclusion body formation and thereby ameliorate the disorder. Honig and co-workers (34) showed that O-methyl threonine, an antagonist of isoleucine, selectively inhibits y chain production in human cord blood, without effect on o( chain synthesis. This result, together with the above mentioned studies on antibiotics (5) and the findings reported here concerning the selective action of polynucleotides and magnesium on ac and @ chain synthesis, respectively, establish a precedent for drug action which could eventually lead to the development of compounds useful in the treatment of these severe and common inherited syndromes.

Acknowledgment-We n-is11 to thank Marion Jacobsen for her excellent technical assistance.

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Harvey F. Lodish and David G. Nathan GLOBIN SYNTHESISβ

AND αRegulation of Hemoglobin Synthesis: PREFERENTIAL INHIBITION OF

1972, 247:7822-7829.J. Biol. Chem. 

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