E M B R Y O N I C H E M O G L O B I N S Y N T H E S I S
93
is hardly possible to review this voluminous literature (Schweet and
Bishop, 1963). It will be useful to recall the general outline of this
process, however, for we shall be preoccupied with regulation of Hb
biosynthesis and its importance for red blood cell differentiation. It
should be remembered that most of this information is based on studies
of the rabbit reticulocyte, a stage of erythropoiesis when probably about
10-20% of the final Hb content is synthesized (Borsook et al., 1962).
The primary amino acid sequence of a given polypeptide subunit of
globin is assembled by a stepwise polymerization, proceeding from the
amino to the carboxyl terminus of the polypeptide chain. A given amino
acid which is to be incorporated into a peptide bond has already been
activated by ATP and transferred to a specific molecule of transfer RNA
(tRNA) appropriate for that amino acid. The amino acid is linked by a
3'-acyl bond to the terminal AMP of the tRNA. A specific region of the
tRNA (probably 3 nucleotides) pairs by hydrogen bonding to a complementary sequence of nucleotides on a template RNA (messenger RNA,
synthesized on a nuclear DNA template). The previously polymerized
amino acid is still attached to the template via its own tRNA. In one
or more enzymatic steps a new peptide bond is formed between the
carboxyl group of the previously incorporated amino acid and the amino
group of the incoming amino acid, producing a nascent polypeptide
extended by one amino acid attached by its carboxyl terminus to a
tRNA which is in turn hydrogen bonded to the messenger template.
The bond between the previously incorporated amino acid and its tRNA
is broken during the process, and this tRNA is no longer tightly bound.
This stepwise addition continues until the polypeptide chain is completed. After completion of the polypeptide chain, it must be released
from the template, generate its tetrameric conformation in association
with other polypeptides, and associate with heme. These latter steps in
the process are still very poorly understood, although some recent
information bearing on aspects of it will be discussed subsequently.
This model of Hb biosynthesis places the information for the primary
sequence solely in the messenger templates associated with the ribosomes. Other factors might also specifically affect the amino acid sequence: the amino acid activating enzymes, tRNA, ribosomes, or peptide
bond-forming enzymes. There is evidence that specification of sequence
may be determined in part by so-called "soluble factors." In this type
of experiment Hb biosynthesis, in vitro, is carried out in a mixed system;
the ribosomes are prepared from reticulocytes of one species and the
soluble portion (100,000 X g, supernatant) is derived from reticulocytes
of another species. The resultant radioactive Hb is then characterized
by any one of a number of procedures, and several experiments of this
type purport to show some Hb characteristic of the species providing
the supernatant may be formed (reviewed by Schweet and Bishop, 1963;
93
is hardly possible to review this voluminous literature (Schweet and
Bishop, 1963). It will be useful to recall the general outline of this
process, however, for we shall be preoccupied with regulation of Hb
biosynthesis and its importance for red blood cell differentiation. It
should be remembered that most of this information is based on studies
of the rabbit reticulocyte, a stage of erythropoiesis when probably about
10-20% of the final Hb content is synthesized (Borsook et al., 1962).
The primary amino acid sequence of a given polypeptide subunit of
globin is assembled by a stepwise polymerization, proceeding from the
amino to the carboxyl terminus of the polypeptide chain. A given amino
acid which is to be incorporated into a peptide bond has already been
activated by ATP and transferred to a specific molecule of transfer RNA
(tRNA) appropriate for that amino acid. The amino acid is linked by a
3'-acyl bond to the terminal AMP of the tRNA. A specific region of the
tRNA (probably 3 nucleotides) pairs by hydrogen bonding to a complementary sequence of nucleotides on a template RNA (messenger RNA,
synthesized on a nuclear DNA template). The previously polymerized
amino acid is still attached to the template via its own tRNA. In one
or more enzymatic steps a new peptide bond is formed between the
carboxyl group of the previously incorporated amino acid and the amino
group of the incoming amino acid, producing a nascent polypeptide
extended by one amino acid attached by its carboxyl terminus to a
tRNA which is in turn hydrogen bonded to the messenger template.
The bond between the previously incorporated amino acid and its tRNA
is broken during the process, and this tRNA is no longer tightly bound.
This stepwise addition continues until the polypeptide chain is completed. After completion of the polypeptide chain, it must be released
from the template, generate its tetrameric conformation in association
with other polypeptides, and associate with heme. These latter steps in
the process are still very poorly understood, although some recent
information bearing on aspects of it will be discussed subsequently.
This model of Hb biosynthesis places the information for the primary
sequence solely in the messenger templates associated with the ribosomes. Other factors might also specifically affect the amino acid sequence: the amino acid activating enzymes, tRNA, ribosomes, or peptide
bond-forming enzymes. There is evidence that specification of sequence
may be determined in part by so-called "soluble factors." In this type
of experiment Hb biosynthesis, in vitro, is carried out in a mixed system;
the ribosomes are prepared from reticulocytes of one species and the
soluble portion (100,000 X g, supernatant) is derived from reticulocytes
of another species. The resultant radioactive Hb is then characterized
by any one of a number of procedures, and several experiments of this
type purport to show some Hb characteristic of the species providing
the supernatant may be formed (reviewed by Schweet and Bishop, 1963;