points to species- and cell-specific differences in
the translation apparatus.
Of particular interest is the in vitro synthesis of
the silk fibroin which, at 350-415 kDa, is one of
the largest known polypeptides. In the rabbit reticulocyte system, the synthesis of the complete
chain takes 85 min. The appearance of discrete
classes of shorter chains indicates that translation
is interrupted at particular sites on the mRNA. In
intact silk-gland cells, the synthesis of the silk fibroin molecule is completed in less than 60 min,
but here too the process is discontinuous. The
320-kDA large fibroin molecule used for web
construction by the spider Nephila claviceps is
also produced discontinuously. The biological
sense of this mode of synthesis is not clear [24].
3.2.1 Aminoacyl-tRNA Synthetases
To become involved in protein biosynthesis, the
amino acids must first be attached to tRNA. For
each amino acid there are normally available several isoacceptor tRNAs, but only one specific
aminoacyl-tRNA synthetase. The synthetases
bind covalently to the uridine in position 8 that is
found in all cytoplasmic tRNAs. In prokaryotes,
the enzyme has subunits of 50-300 kDA and
these are arranged as mono-, di- or tetramers. In
contrast, in mammals, the synthetases for at least
nine of the standard amino acids form 18S multienzyme complexes of about 1 MDa. The synthetases of the other amino acids are found in vitro
as di- or tetramers, but in living cells are probably
also associated with complexes. All mammalian
synthetases have subunits of 50-160 kDa
[47, 82). In Drosophila, synthetase complexes of
more than 1 MDa have also been described. The
functional significance of these complexes
remains a mystery.
The synthetases for glycine, alanine and serine
are particularly active in fibroin synthesis and
have been isolated from the silk glands of Bombyx mori. The glycine- and serine-specific enzymes are dimers of 160 and 124 kDa, and the
alanine-specific enzyme is a monomer of 115 kDa
that is so similar to the tetrameric enzyme of
E. coli, which shows the same specificity, that it
shows immunological cross-reactivity [28, 222,
285]. Using immunological methods, it can be
shown that the mitochondrial and cytoplasmic
phenylalanine tRNA synthetases are very closely
related, even in organisms as far apart as the
chicken and yeast; the two isoenzymes are presumably encoded by duplicated chromosomal
3.2.2 Initiation, Elongation and Termination
81
genes [76]. Although there are as yet no complete
sequences for animal synthetases, it is already
clear that more than one protein super-family is
involved. The aminoacyl-tRNA synthetases also
catalyse the condensation of 2 ATP to Ap,A,
which plays an as yet unknown role in DNA synthesis, cell growth and heat-shock reactions [47].
3.2.2 Initiation, Elongation and Termination
The course of translation consists of three subprocesses, each of which requires specific protein factors; these processes are initiation, elongation
and termination. Many basic studies of protein
biosynthesis have made use ofthe cell-free, rabbit
reticulocyte system. Amongst the invertebrates,
the brine shrimp Artemia salina and the fly Drosophila melanogaster have been especially thoroughly investigated. Translation begins at the first
AUG codon of the mRNA; one exception to this
is the aldolase gene of the malarial agent Plasmodium Jalciparum, which uses the normal stop
codon UAG as a start signal [79]. During initiation in eukaryotes, a 43 S complex first forms
from the small ribosomal subunit and the initiator
tRNA; binding ofthe mRNA then leads to a transient 48 S complex that combines with the large
subunit to produce an active translation complex
of 80 S. The series of protein factors involved in
translation are described below using the recently
agreed terminology of the IUB. The eukaryotic
initiation factors (elF) include polypeptides of
15-220 kDa and are found as monomers (elF-I,
eIF-3A, eIF-5, eIF-5A) , homodimers (eIF-4B),
or complicated molecules with three (eIF-2, elF4, eIF-4F), five (eIF-2B) or eight (eIF-3) different subunits. The phosphorylation of serine or
threonine residues in certain subunits is apparently an important regulation mechanism of
translation [105].
e1F-2 forms a complex with GTP and tRNAr et
that then enables interaction between the mRNA
AUG codon and the small subunit. After formation of the 80 S initiation complex by association
of the large subunit, the GTP is hydrolysed and
the e1F-2 bound to GDP is released. After
exchange of GDP for GTP, e1F-2 once again
takes part in the formation of an initiation complex. Haem deficiency in the rabbit reticulocyte
system causes the subunit elF-2a to be phosphorylated by a specific protein kinase, the haemcontrolled suppressor (HeR), and so to lose the
ability for GDP/GTP exchange. In this way, the
formation of the initiation complex, and thus the
the translation apparatus.
Of particular interest is the in vitro synthesis of
the silk fibroin which, at 350-415 kDa, is one of
the largest known polypeptides. In the rabbit reticulocyte system, the synthesis of the complete
chain takes 85 min. The appearance of discrete
classes of shorter chains indicates that translation
is interrupted at particular sites on the mRNA. In
intact silk-gland cells, the synthesis of the silk fibroin molecule is completed in less than 60 min,
but here too the process is discontinuous. The
320-kDA large fibroin molecule used for web
construction by the spider Nephila claviceps is
also produced discontinuously. The biological
sense of this mode of synthesis is not clear [24].
3.2.1 Aminoacyl-tRNA Synthetases
To become involved in protein biosynthesis, the
amino acids must first be attached to tRNA. For
each amino acid there are normally available several isoacceptor tRNAs, but only one specific
aminoacyl-tRNA synthetase. The synthetases
bind covalently to the uridine in position 8 that is
found in all cytoplasmic tRNAs. In prokaryotes,
the enzyme has subunits of 50-300 kDA and
these are arranged as mono-, di- or tetramers. In
contrast, in mammals, the synthetases for at least
nine of the standard amino acids form 18S multienzyme complexes of about 1 MDa. The synthetases of the other amino acids are found in vitro
as di- or tetramers, but in living cells are probably
also associated with complexes. All mammalian
synthetases have subunits of 50-160 kDa
[47, 82). In Drosophila, synthetase complexes of
more than 1 MDa have also been described. The
functional significance of these complexes
remains a mystery.
The synthetases for glycine, alanine and serine
are particularly active in fibroin synthesis and
have been isolated from the silk glands of Bombyx mori. The glycine- and serine-specific enzymes are dimers of 160 and 124 kDa, and the
alanine-specific enzyme is a monomer of 115 kDa
that is so similar to the tetrameric enzyme of
E. coli, which shows the same specificity, that it
shows immunological cross-reactivity [28, 222,
285]. Using immunological methods, it can be
shown that the mitochondrial and cytoplasmic
phenylalanine tRNA synthetases are very closely
related, even in organisms as far apart as the
chicken and yeast; the two isoenzymes are presumably encoded by duplicated chromosomal
3.2.2 Initiation, Elongation and Termination
81
genes [76]. Although there are as yet no complete
sequences for animal synthetases, it is already
clear that more than one protein super-family is
involved. The aminoacyl-tRNA synthetases also
catalyse the condensation of 2 ATP to Ap,A,
which plays an as yet unknown role in DNA synthesis, cell growth and heat-shock reactions [47].
3.2.2 Initiation, Elongation and Termination
The course of translation consists of three subprocesses, each of which requires specific protein factors; these processes are initiation, elongation
and termination. Many basic studies of protein
biosynthesis have made use ofthe cell-free, rabbit
reticulocyte system. Amongst the invertebrates,
the brine shrimp Artemia salina and the fly Drosophila melanogaster have been especially thoroughly investigated. Translation begins at the first
AUG codon of the mRNA; one exception to this
is the aldolase gene of the malarial agent Plasmodium Jalciparum, which uses the normal stop
codon UAG as a start signal [79]. During initiation in eukaryotes, a 43 S complex first forms
from the small ribosomal subunit and the initiator
tRNA; binding ofthe mRNA then leads to a transient 48 S complex that combines with the large
subunit to produce an active translation complex
of 80 S. The series of protein factors involved in
translation are described below using the recently
agreed terminology of the IUB. The eukaryotic
initiation factors (elF) include polypeptides of
15-220 kDa and are found as monomers (elF-I,
eIF-3A, eIF-5, eIF-5A) , homodimers (eIF-4B),
or complicated molecules with three (eIF-2, elF4, eIF-4F), five (eIF-2B) or eight (eIF-3) different subunits. The phosphorylation of serine or
threonine residues in certain subunits is apparently an important regulation mechanism of
translation [105].
e1F-2 forms a complex with GTP and tRNAr et
that then enables interaction between the mRNA
AUG codon and the small subunit. After formation of the 80 S initiation complex by association
of the large subunit, the GTP is hydrolysed and
the e1F-2 bound to GDP is released. After
exchange of GDP for GTP, e1F-2 once again
takes part in the formation of an initiation complex. Haem deficiency in the rabbit reticulocyte
system causes the subunit elF-2a to be phosphorylated by a specific protein kinase, the haemcontrolled suppressor (HeR), and so to lose the
ability for GDP/GTP exchange. In this way, the
formation of the initiation complex, and thus the