An analysis of arginine codon multiplicity in rabbit hemoglobin

J. Mol. Biol. (1967) 28, 275-280

An Analysis o f Ar0nine Codon Multiplicity in Rabbit Hemoglobin BERNARD WI~.ISBLUM

DeTar~ment of Pharmacology, University of Wisconsin Medical ~chool Madison, Wisconsin, U.S.A.

JosEP~ D. CsmP,Armt, RO~.RTM. Boox De~artmen~of BiochemisSry, UniversiSyof Wisconsin, Madison, Wisconsin, U.S.A. AND In~ituSe for Enzyme Research, Univer~rityof Wiscon~n Madison, Wisconsin, U.S.A. (Received 8 May 1967) Two arginyl tRNA's from yeast (Arg t R N A I and Arg tRNA I I ) are separable b y chromatography. Arg t R N A I which binds to the Escherich/a coZ~ribosomes in response to the m'plets CpGpU§, CpGpC and CpGpA, transfers arginine only into the C-terminal position of the a-chain of rabbit hemoglobin (tryptic peptide ~T17, position 141). Arg t R N A I I , which binds to ~. co~i ribosomes in response to the triplets ApGpA and ApGpG, transfers argln~ue into position 31 of the ~-chain (corresponding to peptide aT4). Codon assignments to positions 31 and 141 of the a-chain, based on the utilization of arglniue from either of the two t R N A fractions, are consistent with known amino acid replacements at these positions, and could have been produced by a change of a single base. l~o labeled arg~n~ue was~ transferred into positlon 92 (corresponding to peptide a T l l ) nor into the two E-chain arg~nlue positions. The lack of labeling of position 92 m a y be due to its specification b y the triplet CpGpG, since neither of the t R N A fractions was bound to ribosomes in the presence of CpGpG. This codon assignment is consistent with the observed replacement of arginine at position 92 b y leueine and glutAmlue in mutant human hemoglobins. The lower level of ~.chalns synthesis precluded a determination of the transfer specificity of E-chain arginine residues. Nevertheless, only the triplet ApGpPu at positions 31 and 41 of the E-chain can be interrelated with observed amino acid replacements, b y a change of a single base. 1. I n t r o d u c t i o n The six codons which have been assigned to the amino acid arglniue fall into two m a i n subgroups: (a) OpGpU, CpGpC, OpGpA and OpGpG; (b) A p G p A and A p G p G (Brimacombe d aL, 1965; Morgan, Wells & Khorana, 1966). Two arginyl t R N A ' s from yeast, Present address: Indian Institute of Science, Department of Biochemistry, Bangalore, India. Present address: Department of Molecular Biophysics, Yale University, l~ew Haven, Conn., U.S.A. § Abbreviations used: CpGpU refers to the trirlbonueleoside diphosphate-cytidylyl (3"--5") guanylyl (3"-->5~) uridine; the other triplets are named according to this convention; tRNA, transfer RIGA. 275

276

B. W E I S B L U M , J. D. C H E R A Y I L , R. M. B e C K A N D D. 8 0 L L

Arg t R N A I and Arg t R N A H have been separated b y SSll et al. (1966) and studied with respect to triplet binding specificity. Arg t R N A I was bound to Escherichia coli ribosomes in the presence of subgroup a triplets and Arg t R N A I I was bound in response to subgroup b triplets. Multiplicity of t R N A has also been studied in E. cell, where two separable types of leucyl-tRNA corresponding to tw~o major subgroups, transferred leucine into distinct positions in rabbit hemoglobin (Weisblum, Gonano, yon Ehrenstein & Benzer, 1965). Because relatively little is known about the generality of this phenomenon, or about the relative utilization of various multiple codons in the synthesis of natural proteins, the present studies on rabbit hemoglobin synthesis were undertaken. As will be shown, the two yeast arginyl-tRNA's transfer arginine into different positions in rabbit hemoglobin in a way which suggests t h a t the three arglnlne residues of the rabbit ~-chain correspond to three different codons. 2. M a t e r i a l s a n d M e t h o d s

sRNA, prepared from yeast, was fractionated by chromatography on DEAE-Sephadex, and charged with arginine as described by $611 et al. (1966). Thus Arg tRNA I (50 A2e0 units) charged with 800,000 cts]min [3H]arginine, and Arg tRNA I I (100 A2so units) charged with 100,000 cts]min [14C]arginine were prepared. Uniformly labeled [14C]arginine, specific activity 220/~c]mole, was obtained from the New England Nuclear Corporation, Boston, Mass., U.S.A. Generally labeled [3H]arginine, specific activity 2,300 ~c/~mole, was obtained from the International Chemical & Nuclear Corporation, City of Industry, Calif., U.S.A. Transfer of arginine from the charged tRNA "fractions into hemoglobin was performed using a reaction mixture, volume 10 ml., according to the method previously described for leucine incorporation (Weisblum et al., 1965). Twenty amino acids in unlabeled form, 2 pmoles each, were present during the transfer. After the incubation was completed, carrier hemoglobin (100 mg) was added to the incubation mixture and the ribosomes were removed by centrifugation at 100,000 g for 2 hr. Globin, prepared from the supernatant fraction of the previous step, was digested with trypsin. The tryptic peptides were separated by paper electrophoresis in a tank device (2 hr, 45 v/cm, 150 n~). The buffer used contained 50 ml. pyridine, 50 ml. glacial acetic acid, 1900 ml. water (pH 4.6). The peptide nomenclature of Diamond & Braunitzer (1962) was used. For determination of radioactivity, the paper was dried and cut up into 1-cm zones which were eluted with acetic acid : water (1 : 100, v/v). The eluates were dried, dissolved in 0.1 ml. water-methanol (1 : 1, v/v) taken up in 10 ml. scintillator solution and counted with a Packard liquid-scintillation counter at ambient temperature. In collecting the data presented in Fig. 1(b) and (c), counting conditions were used which permitted simultaneous determination of 3H and 14C in the sample. The scintillator solution contained 10 g 2,5diphenyloxazole, 0-35 g 1,4-bis (2-(5 phenyloxazolyl)-benzene, and 300 g naphthalene, in 3000 ml. dioxane. 3. R e s u l t s

There are five arginine residues in rabbit hemoglobin; three are in the ~-ehain and two are in the E-chain (Naughton & Dintzis, 1962; Colombo & Baglioni, 1966). The tryptic peptides containing these residues are well separated b y paper electrophoresis at p H 4.6 (Colombo & Baglioni, 1966). R a b b i t hemoglobin, prepared b y incubating intact reticulocytes with [14C]arginine, provided a set of reference peptides (see Fig. l(a)), and the presence of arginine in these peptides was verified using the Sakaguchi reaction according to the technique described b y Easley (1965). [3H]Arginine charged to t R N A I and [14C]arginine charged to t R N A I I were transferred into rabbit hemoglobin in the same reaction mixture. This t y p e of experimental design is preferable to two single transfer experiments since it provides evidence

ARGININE CODON M U L T I P L I C I T Y IN HEMOGLOBIN

277

concerning the concurrent performance of two tRNA's for the same amino acid bearing different coding specificities. The data presented in Fig. l(b) and (e) pertain to the specificity of [SH]-and [l~C]arginine-transfer into various positions in rabbit hemoglobin.

200

"'~ . "' ~

I

I

I

I

il

L'4C.IArginineincorporated by intoct reticulocytes

8 150 .a.-~ o ~ ioo

".~

/l

A/3T4cxTll.e,2[ ~TII

;-=:----I(b)

I

I0

I "

20

250 "=

o ~ IS00

~D

~ tO00"

SO

E~

Origln

ol ~ ~ FI I0 20 I50F ~4C]Arginlne •

I

u

40

[3H~Arglnine from tRNAT_

200(

o ~

I'""I

30 o:T17

0

I 40

-'~

30

ro,,RNA II ' T4

10Ols/

I 30

II ".J

I0

• ~,,,,~,..,_, _, / \

20

30

_.

40

50

Cm

~IG. 1. Specffieity of arginino t~ansfer into different positions of rabbit hemoglobin. [eH]- and [z~C]arginine were transferred from Arg tRNA I and II, respectively, into rabbit hemoglobin as described in Materials and Methods. The pattern of [z4C]argin~e incorporation by intact reticulocytes is presented in (a). [3HI- and [z4C]arginine incorporation, by coil-freetransfer into various positions, is presented in (b) and (c), respectively.

The labeling pattern of tryptie peptides obtained from the double-label transfer was compared with the pattern obtained from the tryptic digest of [14C]arginlnelabeled hemoglobin synthesized by intact reticulocytes. (cf. Fig l(a) with (b) and

(e).) At least four significant features m a y be noted. (1) t R N A I preferentially transfers its arglnlne (labeled with 3H) into peptide ~T17 (position 141); (2) t R N A I I preferentially transfers its arginine (labeled with z4C) into peptide ~T4 (position 31); (3) neither t R N A I nor tRNA I I transfers its arginine into peptide ~ T l l or peptide ~ T l l ~ 12 (position 92);

278

B. W E I S B L U M ,

J. D. C H E R A Y I L ,

R. M. B O C K A N D D.

SOLL

(4) neither tRNA I nor tRNA H transfers signi~eant amounts of argln~ne into jgT3 or ~f4 (positions 31 and 41, respectively). Labeled material present in fractions 37 and 38 is present in all three analyses. ~rom other experiments (data not shown), this corresponds to free arg~nlne. The composition of the 3H-labeled material in fraction 40 is unknown. It may have been formed during storage for 48 hours at --20°C, because it was not seen in a previous fractionation of the same tryptic digest. Thus, on the basis of the ~ransfer data obtained in these experiments and the binding specificity of the tRNA's used, the three argln~ne residues of the ~-chain correspond to three different codons in the messenger. Arginine at position 31 corresponds to an ApGpPu codon, arginlne at position 141 corresponds to CpGp(U, C, A), and argln~ne at position 92 corresponds to neither of these codon assignments. 4. D i s c u s s i o n

For each of the arglnlne residues considered, at least one amino acid replacement has been observed which could be uniquely accounted for by a single base change in the postulated codon. It is assumed that the codon for a given position is most likely to be that one which requires the fewest nucleotide changes to account for observed replacements. The interpretation of this analysis, based on replacementand ar~n~ne-transfer data, is tabulated in Fig. 2.

~-ohain

31

92

141

Arg

Arg

Arg

A~

CGG

CG~

]A~

CUG/~CAG Leu Gin

& His

Lys

~-chain

31

41

Arg

Arg

A~ I AA~

AG~ l AC~

Lys

Thr

~G. 2. Summary of arginine codon assignments in rabbit hemoglobin based on amino acid transfer, and replacement-data.

The most marked difference between the transfer specificities of the two tRNA's is with respect to peptide ~T17 (fraction 28). Since tRNA I with the specificity C~Gp(U, C, A) transfers into this position, we tentatively assign the codon CpGp(U, C, A) for arglnlne at this position. The only position available for comparison is number 141 in the E-chain, which contains histidine in all known hemoglobins. Histidine is represented by the codons CpAp Pyr. Therefore, the codon assignment for argln~ne may be refined further to CpGp Pyr, which is compatible with the observed replacement at position 141 being caused by a transition mutation, CpGp Pyr (arg~n~ne) ~ CpAp Pyr (ins"tidine). Some 3H-label is found in peptide ~T4. This may be due

ARGININE

CODON

MULTIPLICITY

IN HEMOGLOBIN

279

to tRNA impurity, or to a different peptide component which fractionated with T4 on electrophoresis. Transacylation cannot be completely ruled out, since rabbit reticulocyte-activating enzymes can partially charge yeast tRNA with arglu~ue (Weisblum, unpublished results). The ratio SH~14Cfor fraction 17 is 2.8 compared to the input ratio of 8.0. Thus there is at least a threefold enrichment of 14C at this position. All known hemoglobins contain arg~nine in position S1 of the ~-chain (corresponding to peptide roT4), whereas lamprey myoglobin contains lysine in a homologous position (Braunitzer, 1966). The only possible single-step mutation interrelating lysine and argluine would be: ApApPu (lysine) < ~ ApGpPu (arginine); i.e. the arglniue specified by tRNA II. There is an apparent absence of either SH- or 14C-label from peptide ~Tll or peptide ~Tll ~- 12 (position 92). This cannot be attributed to lack of resolution, since, first, the fractionation system used consistently separates ~Tll and ~T17 completely. Even if the separation of the two peptide bands were less than~l cm, mTll would have appeared as a shoulder on the right side of roT17, as in Fig. l(a). The results shown in Fig. l(a) demonstrate the least separation observed; often two completely separated peaks are found. Second, peptides ~Tll and ~T17 were located by stalnlng with the Sakaguchi test and were found to be completely separated. The peptides were eluted and counted, and no radioactivity was detected in ~Tll. The absence of label could be explained by examlning mutational replacements at the corresponding position in human hemoglobin, which also contains arglulne at position 92. Hemoglobins Chesapeake and Capetown, however, contain leucine and glutamlne, respectively, at this position (Clegg, Naughton & Weatherall, 1966; Botha, Beale, Isaacs & Lehmann, 1966). These data can be reconciled if we assume the following single-step mutational events:

#cu

A

A /(Leucine) CGa~ (Glut~ml-e) Since there was no CpGpG-dependent binding activity in the tRNA fractions used in these experiments, it is probable that the purine involved is G. This hypothesis can be tested directly using a tRNA specific for CpGpG. The pattern of OpGp-codon recognition found in yeast is not unique. In addition to tRNA specific for CpGp(U, C, A), guinea pig liver also contains tRNA specific for OpGpPu (Caskey & Nirenberg, personal communication). Such a tRNA could also be useful in discriminating between A and G in the third position. In view of previous experience with cell-free synthesis of hemoglobin (yon Ehrenstein Weisblum & Benzer, 1963), the failure to observe arg~n~ne incorporation into peptides jgT3 and JgT4 is not too surprising. In these previous studies, leucine, alanine and cysteine were transferred into hemoglobin from charged sRNA, and a higher level of incorporation into the ~-chain relative to the jg-chain was consistently observed. This is especially significant for cysteine incorporation, because there is only one residue in each chain at roughly the same position relative to the carboxyl terminus. Colombo & Baglloni (1966) have recently proposed a mechanism to explain the disparity between the relative amounts synthesized in v~tro. In the absence of any significant incorporation into ~9-chain residue 31 (peptide jgT3), we can only infer its eodon specificity from

280

B. WEISBLUM, J. D. CHERAYIL, R. M. BeCK AND D. SOLL

that of residue 31 of the ~-chain, also arginlne, for which tRNA I I was found to be specific. Presumably, therefore, it has a s~milar codon specificity, i.e. ApGpPu. Finally, the arglnlne at position 41 of the fl-chain (peptide ~r4) is homologous to threonine in the ~-chain. The only single-step mutation interrelating threonine and arglulne would be: ApGpPu~ ~ApCpPu, so that residue 41 could also correspond to tRNA H. A transfer reaction in which more extensive fl-chain synthesis occurred would permit testing this codon assignment experimentally. A summary of the arginine codon assignments in rabbit hemoglobin is tabulated in Fig. 2. Some of the information about arginine codons presented above could have been determined by replacement data solely. The present investigation illustrates how tRNA's of known triplet binding specificity can give additional information about the possible eodon classes corresponding to a particular residue. This is especially useful in relation to the three six-fold degeneracies in the genetic code for leueine, arg~nine and serine, and may find application in assigning codons to residues in other proteins which can be synthesized in vitro and for which extensive replacement data are not available. We thank Dr Walter Fitch for placing his extensive compilation of amino acid replacements at our disposal, and Mrs Darlene Olson for expertly assisting us in this work. For critically reading the manuscript we are also grateful to I)rs L. A. Fahien and J. H. Subak-Sharpe. This work was supported by research grants from the National Science Foundation (GB3080) and from the U.S. National Institutes of Health (GM12395 and FR00214). REFERENCES Bo~ha, M. C., Beale, D., Isaacs, W. A. & Lehmann, H. (1966). Nature, 212, 792. Braunitzer, G. (1966). J. GeIL Phy~iet. 67, suppl. 1, 1. Brimacombe, R., Turpin, J., Nirenberg, M., Leder, P., Bernfield, M. & Jaouni, T. (1965). Prec. Nat. Acad. ~ci., Wash. 54, 954. Clegg, ft. B., Naughton, M. A. & Weatherall, D. J. (1966). J. Mot. Biol. 19, 91. Colombo, B. & Baglioni, C. (1966). J. Mot. BIOL 16, 51. Diamond, L. K. & Braunitzor, G. (1962). Nature, 194, 1287. Easley, C. W. (1965). Biochim. biophys. Acta, 107, 386. yon Ehrenstein, G., Weisblum, B. & Benzor, S. (1963). Prec. Nat. Acad. Sci., Wash. 49, 669. Morgan, A. R., Wells, R. D. & Khorana, H. G. (1966). Prec. Nat. Acad. Sci., Wash. 56, 1899. Naughton, H. & Dintzis, H. M. (1962). Prec. Nat. Acad. Sci., Wash. 48, 1882. SSU, D., Jones, D. S., Ohtsuka, E., Faullrner, R. D., Lohrmann, R. D., Hayatsu, H., Khorana, H. G., Cherayil, J. D., Hampel, A. & Beck, R. M. (1966). J. Mot. Biol. 19, 556. Weisblum, B., Gonano, F., von Ehrenstein, G. & Benzer, S. (1965). Prec. Nat. Acad. ~ci., Wash. 53, 328.