(Received for publication, December 16, 1996)
From the Department of Molecular Biology, Faculty of
Biology, Moscow State University, 119899 Moscow, Russia and
§ Institute of Protein Research, Russian Academy of
Sciences, 142292 Pushchino, Moscow Region, Russia
Globin synthesis in a wheat germ cell-free
translation system was performed in the presence of
[3H]hemin and [35S]methionine to
determine the minimal length of the nascent ribosome-bound globin chain
capable of heme binding. Nascent polypeptides of predetermined size
were synthesized on ribosomes by translation of truncated mRNA
molecules. Analysis with the use of sucrose gradient centrifugation and
puromycin reaction revealed that the ribosome-bound N-terminal
-globin fragments of 140, 100, and 86 amino acid residues are
capable of an efficient heme binding, whereas those of 75, 65, and 34 amino acid residues display a significantly weaker, or just
nonspecific, affinity to heme. This indicates that the ribosome-bound
nascent chain of 86 amino acid residues has already acquired a spatial
structure that allows its interaction with the heme group or that heme
attachment promotes the formation of the proper tertiary structure in
the ribosome-bound nascent peptide. In any case the cotranslational
folding of globin is suggested.
The mechanism by which the growing polypeptide chain acquires its native conformation has been discussed in many recent reviews (1-5). Historically, most protein-folding studies were based on the analysis of protein refolding in vitro. These studies have provided basic insights into the principles and mechanisms governing the folding of polypeptides into compact three-dimensional structures (6-9). However, the in vivo folding is assumed to proceed cotranslationally (10-15). Evidence suggesting the cotranslational protein folding has come from experiments that demonstrated (i) the enzymatic activity of the growing polypeptide on the ribosome (16-21), (ii) the formation of correct epitopes able to bind corresponding conformational antibodies (22), and (iii) the formation of correct disulfide cross-bridges in the growing nascent chains (23-28).
Previously we reported data indicating that heme attachment to the
globin chains may proceed cotranslationally (29). Analysis of globin
synthesis in cell-free extracts of rabbit reticulocytes carried out in
the presence of 3H-labeled hemin revealed the presence of
[3H]hemin in the polyribosome fraction synthesizing
globin chains. The addition of puromycin resulted in the release of
both [3H]hemin and 14C-labeled leucine
polypeptide from the polyribosomes. The data obtained indicated
cotranslational heme binding to the nascent globin chains and thus to
the cotranslational folding of the globin molecule since heme binding
in the case of globin molecules is a function of the definite protein
structure (30, 31). However, the possibility of globin tetramer
assembly on the ribosome in those experiments could not be excluded.
Cotranslational trimerization of the retrovirus cell attachment
protein, 1, has been demonstrated recently (32), and thus the
assembly of the nascent globin chains could as well be the case. If
this was the case, the previously obtained data could be alternatively
explained by the presence of complete globin chains (with labeled
hemin) associated with the nascent chain.
To rule out this possibility, we have used the wheat germ cell-free
translation system, which does not contain endogenous globin molecules.
We have also performed the translation experiments with -globin
synthesis, which, in contrast to
-globin, does not form the tetramer
structure (31). We have found that
-globin is capable of heme
binding during its synthesis on the ribosome. In addition, we have
demonstrated that incomplete
-globin molecules of 140, 100, and 86 amino acid residues (lengths are given excluding the first initiator
methionine) are capable of cotranslational heme binding with an
approximately equal efficiency, whereas polypeptide chains of 75, 65, and 34 amino acid residues display a significantly weaker, or just
nonspecific, affinity to heme. This shows that the nascent chain of 86 amino acid residues possesses a spatial structure that allows its
interaction with the heme group or that the heme attachment promotes
the formation of the proper tertiary structure of the growing
polypeptide on the ribosome. Hence, the cotranslational folding of
globin molecule is suggested.
The plasmid
PHST101 (rabbit -globin subclone PSP64) containing
-globin
cDNA under control of the SP6 promotor was the gift of Professor J. Ilan, Case Western Reserve University. The transcription reaction was
carried out according to Gurevich et al. (33) in 500 µl
(total volume) of 80 mM HEPES-KOH buffer, pH 7.5, containing 16 mM MgCl2, 2 mM
spermidine, 20 mM dithiothreitol
(DTT),1 3 mM ATP, 3 mM GTP, 3 mM UTP, 3 mM CTP, 15 µl
(375 units) of RNasin (Pharmacia Biotech Inc.), 25 µg of
BamHI-linearized DNA template, and 2000 units of SP6 RNA
polymerase (Fermentas). The reaction was carried out at 37 °C for
2.5 h and stopped by phenol/chloroform extraction. The transcript
was purified by LiCl precipitation and washed with 70% ethanol (34).
An aqueous solution (3.44 mg/ml) of the transcript was used in
translation experiments.
Cell-free translation of the
-globin mRNA was performed using wheat germ extract as described
by Clemens (35). The reaction mixture contained 20 mM
HEPES-KOH buffer, pH 7.5, 3 mM
Mg(CH3COO)2, 100 mM
KCH3COO, 2.5 mM DTT, 1.3 mM ATP,
0.25 mM GTP, 50 µM spermidine, 16 mM creatine phosphate, 40 µg/ml creatine phosphokinase.
The concentration of mRNA was 100 µg/ml. The specific
radioactivities of [35S]methionine and
[3H]hemin used in the experiments were 1 and 1.2 mCi/ml,
respectively. [3H]Hemin was prepared by the hot tritium
bombardment technique (36) as described previously (29). The final
reaction volume was 100 µl. The translation was carried out at
24 °C. After 25 min of incubation, the reaction mixtures were
subdivided into two equal portions. Puromycin was added to a final
concentration of 1.5 mM to one of the portions, and the
incubation of both portions was continued for an additional 10 min.
To produce
-globin peptides of predetermined lengths in the cell-free system,
the
-globin mRNA was digested with RNase H in the presence of
corresponding antisense oligodeoxyribonucleotides before translation.
The following 20-mer oligodeoxyribonucleotides were used to produce the
truncated mRNA encoding for the N-terminal
-globin fragments
with the lengths indicated as numbers of amino acid residues:
34,
5
-GTCTTGGTGGTGGGGAAGCC-3
;
65, 5
-TGGCCCACGGCCTTGGTCAG-3
;
75,
5
-GTAGACAGGGCGCCGGGCAG-3
;
86, 5
-ACCCGCAGCTTGTGCGCGTG-3
;
100, 5
-ACCAGCAGGCAGTGGGACAG-3
;
140,
5
-TCCCAGGCTCCAGCTTAACG-3
.
10 µg of full-sized globin mRNA was incubated at 37 °C with a
50-fold molar excess of a complementary 20-mer oligodeoxyribonucleotide and 100 units of RNase H from Escherichia coli. The reaction
was carried out for 1 h in 40 mM Tris-HCl buffer, pH
7.6, containing 1 mM DTT, 1 mM
MgCl2, and 30 mg/ml bovine serum albumin. Truncated globin
mRNAs obtained after digestion were used in the translation experiments (Fig. 1). Completeness of RNA digestion was
controlled by 5% polyacrylamide gel electrophoresis in the presence of
7 M urea. To check the specificity of RNase H action,
3-ends of the truncated RNA molecules were determined, and the
truncated
86 mRNA molecule was sequenced using the Pharmacia RNA
sequencing enzyme kit according to the procedure described in Ref. 37. Production of globin peptides of the defined lengths after translation of the truncated mRNAs in the cell-free system was controlled by
electrophoresis as described (38).
Polymerase Chain Reaction (PCR)
PCR fragments were
generated using the oligonucleotides 5-GATTTAGGTGACACTATAGAATACA-3
as
the downstream primer and 5
-GTCGCTGAGAGTAGACAG-3
as the upstream
primer. The plasmid pHST101 was linearized with BamHI and
then amplified using Taq DNA polymerase by 30 cycles of PCR
consisting of 1 min at 94 °C, 1 min at 40 °C, and 1 min at
72 °C each.
Sucrose gradient centrifugation was used to analyze the incorporation of radiolabeled methionine as well as the binding of hemin to the nascent globin peptides retained in the ribosome fraction during globin synthesis. To avoid contamination of unbound [3H]hemin and [35S]methionine, 50-µl aliquots of the translation mixture were washed before sucrose gradient centrifugation with 2 ml of 10 mM HEPES-KOH buffer, pH 7.6, 100 mM KCH3COO, 10 mM Mg(CH3COO)2, 1 mM DTT, and 0.1 mM EDTA on the Centricon C30 (Amicon Inc.) microconcentrator (30 min, 2000 × g, 4 °C). After ultrafiltration, 100-µl aliquots of the washed translation mixture were layered on the top of linear 0.5-1.5 M sucrose gradients in 10 mM HEPES-KOH buffer, pH 7.6, 100 mM KCH3COO, 10 mM Mg(CH3COO)2, 1 mM DTT, and 0.1 mM EDTA. Centrifugation was done for 2.5 h at 41,000 rpm in a Beckman SW 41 rotor at 4 °C. The gradients were pumped from the bottom, and absorbance at 278 nm was continuously recorded. 400-µl fractions were collected, and the radioactivity was counted.
It was shown earlier that globin molecules are capable of
cotranslational binding of heme in a homologous rabbit reticulocyte cell-free system (29). Here we used the same methodology but with the
wheat germ cell-free system. The question was whether the individual
globin molecule (- or
-chain) is capable of cotranslational heme
binding. For the in vitro translation in a wheat germ system we chose the
-globin that has a 10-fold stronger ability to bind the
heme group as compared with the
-globin (30, 39) and, in contrast to
-globin, does not form the tetramer structure (31).
Fig. 2 shows that [3H]hemin is detected in
the ribosome fraction translating -globin mRNA. The addition of
puromycin results in the release of a major portion of both
[3H]hemin and 35S-labeled methionine
polypeptide. This indicates that the heme is attached to the nascent
-globin chain on the ribosome.
Incomplete
The question is whether the heme retention can be observed on incomplete nascent globin chains, especially after both histidine imidazole groups of the E and F helices necessary for heme attachment have emerged from the ribosome. Taking into account that the translating ribosome may protect about 15-40 amino acid residues of the nascent peptide (40-45), it could be assumed that only the nascent peptides of approximately 100 amino acid residues and longer were capable of binding the heme (29).
To answer this question, the method of translation arrest by antisense
oligodeoxynucleotides was chosen. The translation of mRNAs,
truncated by RNase H in the presence of complementary oligonucleotides, results in nascent polypeptides of predetermined lengths attached to
the ribosome as was reported previously (46). It was also reported that
the dominant RNase H cut occurs at the RNA 5-end in the RNA
oligodeoxyribonucleotide complex in conditions of complete hydrolysis
(47). Thus, the length of nascent peptide is predictable (48, 49). We
started with the globin mRNA lacking the last 3
-terminal coding
triplet. Sucrose gradient centrifugation analysis revealed that such an
incomplete nascent globin peptide (
-globin 140; the length here does
not include the first initiator methionine) is capable of heme binding
during translation in the wheat germ system (not shown). Using the same
approach, we found that an incomplete globin peptide of 100 amino acid
residues (
-globin 100) is also capable of cotranslational heme
binding (Fig. 3A). Surprisingly, the same has
been demonstrated for a shorter globin peptide of 86 amino acid
residues lacking the heme-binding histidine residue at position 87 (Fig. 3B). Determination of the 3
-end and sequencing of the
86 mRNA showed slight heterogeneity of the
86 mRNA 3
-end
(not shown). To prove that an
-globin peptide of 86 amino acid
residues is capable of heme binding, we performed the experiments with
86 mRNA obtained after SP6 transcription of a PCR-generated
template containing SP6 promotor and the corresponding part of the
-globin coding sequence. Results obtained in these additional
experiments proved the initial observation (Fig. 3C).
Shorter
As
shown previously in in vitro experiments, the proteolytic
fragment of the -globin molecule comprising residues 31-104 is
capable of binding the heme group (50). The same phenomenon has been
also demonstrated recently for the mini-myoglobin polypeptide fragment
(residues 32-139) (51). To determine the length of a globin chain
sufficient and necessary for cotranslational heme binding, we
translated truncated
-globin mRNAs (Fig. 1) and produced shorter
nascent peptides of 75, 65, and 34 amino acid residues. Incorporation
of radiolabeled methionine as well as hemin into the nascent globin
peptides was controlled after sucrose gradient centrifugation of the
wheat germ translation system as described above. In contrast to the
experiments with longer polypeptides (Fig. 3, A and
B), we found much lower incorporation of
[3H]hemin either into the ribosome-bound polypeptides of
34 (not shown), 65, or 75 amino acid residues (Fig. 3, D and
E).
Since all polypeptides under investigation have an equal number of
methionine residues (at positions 1 and 33), the
[3H]hemin/[35S]methionine ratio can be used
as a measure of the efficiency of heme binding to the nascent
polypeptides of various lengths. Fig. 4 presents the
summary of puromycin effects on the release of nascent
35S-labeled methionine polypeptide and
[3H]hemin from ribosomes after the translation of
full-length and truncated mRNAs. Some amounts of hemin found
attached to the shorter peptides can probably be attributed to a
nonspecific heme adsorption. Alternatively, one can speculate that heme
begins to dock to the globin polypeptide chain very early during its
synthesis when the first amino acid residues competent for heme binding
appear from the peptidyltransferase center, but at these stages the
heme binding is not strong enough to form a stable heme-globin
complex.
A number of experimental findings suggest cotranslational protein
folding (16-28, 32, 52). Thus, the possibility of cotranslational ligand binding cannot be excluded. Studies of biosynthesis of protein
D1 of the membrane-bound chloroplast reaction center directly indicated
the cotranslational binding of chlorophyll to an incomplete D1 molecule
(53, 54). Cotranslational binding was also assumed to take place for
the heme group in the case of globins (29, 55). It is well known that
the detachment or displacement of heme groups is accompanied by
denaturation of hemoglobin, whereas the addition of the heme group to
apohemoglobin or apomyoglobin promotes the formation of the native
structure of the molecules (30, 56-58). It was shown that
mini-apo--globin (residues 31-104) or mini-apomyoglobin (residues
32-139) reconstituted with natural heme preserved conformations
similar to those in the whole molecules (50, 51). Moreover, it was
evident that the heme orientation in the pocket and the coordination
state of the ferrous iron in the mini-globins are just the same as in
the whole molecules (51). On the grounds of these data we suggest that
heme binding to the nascent globin chains can be used as a test of
cotranslational folding of globins. The aim of this work is to
determine the length of the nascent globin chain on which heme
attachment occurs during translation.
We have demonstrated an equally efficient cotranslational incorporation
of [3H]hemin into nascent globin chains of 140, 100, and
86 amino acid residues (as well as into the full-length molecule). The
fact that puromycin (known to release ribosome-bound peptides) causes the release of both 35S-labeled methionine polypeptide and
[3H]hemin from ribosomes (Figs. 2 and 3) indicates that
either the nascent peptide of 86 amino acid residues possesses a
spatial structure allowing its proper interaction with the heme group or the heme attachment promotes the formation of the proper tertiary structure. Hence, the cotranslational formation of the spatial structure of globin at the early stages of its synthesis is likely. It
was recently shown that chemically synthesized peptides with chymotrypsin inhibitor-2 growing from the N terminus acquire the three-dimensional structure in vitro while achieving the
length of 62-63 amino acid residues (59). We believe that the same occurs in the case of nascent 86 (and longer) globin polypeptides growing on the ribosome.
To illustrate our results, we present wire frame models of the well
known three-dimensional structures of the human deoxyhemoglobin -chain (Fig. 5A and Ref. 60) and the
incomplete
86-globin chain (Fig. 5B). The
86-globin
model was produced from the crystal structure of
-chain by skipping
the C-terminal residues. The length of the incomplete
86 molecule in
the model is the same as the lengths of nascent peptides in our
experiments. There are a number of amino acid residues in the
-globin molecule known to be involved in the formation of contacts
with the heme group. Among them are Met-32, Tyr-42, Phe-43, His-45,
Phe-46, Lys-61, Val-62, Ala-65, Leu-83, Leu-86, Leu-91, Val-93, Asn-97,
Phe-98, Leu-101, Leu-136, and two heme-binding His residues, His-58 and His-87 (60). Our results show that the nascent peptide of 86 amino acid
residues lacking the heme-binding histidine residue at position 87 is
capable of heme binding. This may indicate that the contacts provided
by the remaining residues are sufficient for specific heme binding.
Eleven residues involved in heme binding remain in the incomplete
polypeptide of 86 amino acid residues (Fig. 5B). Since the
incomplete nascent peptide of 75 amino acid residues does not bind the
heme group so efficiently (Fig. 3D), it can be speculated
that the leucine residues at positions 83 and 86 provide the necessary
contacts, thus forming part of the heme pocket and allowing the
incomplete nascent chain of 86 amino acid residues to bind the heme
group quite effectively.
If this is the case, all the C-terminal sections of the polypeptide must appear from the ribosome and form E and F helices. It presumes that a nascent polypeptide chain can fold immediately at the peptidyltransferase center of the ribosome and no intraribosomal tunnel exists, as was indicated previously (61-63).
The alternative case could be that a ribosomal tunnel or channel hides the C-terminal section of a growing polypeptide (64, 65). Then the nascent ribosome-bound globin peptide of 86 amino acid residues should be able to bind hemin either without F helix, if just 15 amino acid residues are hidden (41, 42), or without both E and F helices, if 30-40 amino acids are accommodated within the ribosome (39, 42, 44). The latter seems unlikely. Rather, the shielding of the C-terminal part of the growing nascent peptide can now be explained by nascent polypeptide-associated complex binding (63, 66), known to protect about 30 C-terminal amino acid residues from the proteolysis. As nascent polypeptide-associated complex cycle of binding and release was proposed (63), it can be speculated that nascent polypeptide-associated complex is released from the nascent peptides when it acquires the three-dimensional structure.
Taking into account all the facts and considerations mentioned above, we argue that the globin polypeptide chain begins to correctly fold with the participation of heme rather early during its elongation on the ribosome.
We thank Dr. V. Kolb for critical comments
and discussion of the manuscript. We are grateful to Drs. O. Denisenko
and L. Ryabova for helpful advice in translation experiments and Drs.
V. Ksenzenko and N. Kholod for help in RNA sequencing. We thank Dr. O. Kurnasov for help in preparation of -globin mRNA, Dr. N. Chichkova for the gift of RNase H, and Dr. M. Wiedmann for the gift of
the PCR primers.