Biotechnol. Prog.

1996, 12, 417−422 417

Topical Papers
Problems of Translating Heterologous Genes in Expression
Systems: The Role of tRNA
David W. E. Smith
Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611

tRNA can have large effects on the expression and overexpression of heterologous
genes in microbial expression systems through reduced translation and errors in amino
acid sequences of protein products. Examples are given of large effects on gene
expression related to tRNA content and to tRNA base modifications, both of which
differ in heterologous expression systems compared to the cells from which the genes
originally came. tRNA should be of greater concern in the expression of heterologous
genes.

Contents tRNA Function in Protein Synthesis

Introduction 417
tRNA Function in Protein Synthesis 417
tRNA Content of Cells: Adaptation for 417
Protein Synthesis
Quantative Effects of tRNA Content on 417
Translation
Qualitative Effects of tRNA Content on 418
Translation
Modifications of tRNA Bases 419
Position 34 419
Position 37 419
Qualitative Effects of tRNA 420
Modifications on Translation
Quantitative Effects of Modifications 420
on Translation Based on tRNA Choice
Problems and Opportunities 420
Conclusions 421
Introduction
Expression of heterologous genes in microbial expres-
sion systems depends on components of the system,
including the protein synthetic machinery (Goeddel,
1990). This article shows that translation is sometimes
a problematic aspect of gene expression, with the tRNA
of expression systems reducing gene expression and
introducing errors into protein products. Examples are
provided of large effects of tRNA, and a major purpose
of this review is to remind workers of the importance of
tRNA in gene expression. Often gene expression appears
to be satisfactory, and such problems as occur escape
detection.
Heterologous genes are commonly expressed as high
expression fractions of total protein synthesissas much
as 40% of the total protein synthesis of expression
systems (e.g., Goeddel, 1990; Alldread et al., 1992). This
is several times higher than the expression fraction of
any homologous gene product in microbial cells and is
called “overexpression”. It increases specific demands on
the protein synthetic machinery of expression systems.
S8756-7938(95)00056-7 CCC: $12.00 © 1996 American Chemical Society and American Institute of Chemical Engineers
The tRNA species are the translators of the genetic
code, with affinities or recognition sites for aminoacyla-
tion by the aminoacyl-tRNA synthetases, for codons of
mRNA, and for ribosomes and initiation and elongation
factors. (Accurate translation also depends on the ami-
noacyl-tRNA synthetases, with their specificity for amino
acids and tRNA species.) Figure 1 is a diagrammatic
illustration of tRNA functioning and cycling in transla-
tion. tRNA function in translation is more complex than
reading a linear sequence of information by a uniformly
performing family of decoding molecules. Factors influ-
encing tRNA function include codon context, tRNA
interactions on ribosomes, and tRNA efficiency and
leakiness in coding, miscoding, stop codon read-through,
and frameshifting.
There are at least two important respects in which
tRNA of expression systems differs from tRNA of the cells
of origin of foreign genes: (1) the content of tRNA species
and (2) the enzymatic modifications of tRNA bases. Each
can affect translation quantitatively and qualitatively,
with examples provided below in which large percentages
of protein products are affected.
tRNA Content of Cells: Adaptation for Protein
Synthesis
The cellular content of tRNA species often correlates
positively with the codon bias of mRNAs for the major
homologous protein products (Hatfield et al., 1992). This
correlation is common in microorganisms, and it is an
aspect of differentiation of specialized cells of higher
organisms that synthesize unusual proteins in large
amounts. Such tRNA contents have been described as
adapted, modulated, or specialized for protein synthesis.
Quantitative Effects of tRNA Content on Trans-
lation. There is disagreement about whether limiting
tRNA can have a major role in orchestrating or otherwise
regulating translation in homologous cells. On one hand,
the existence of adapted tRNA contents and observations
that elongation of peptides proceeds at variable rates
have been interpreted as evidence for translational
regulation by tRNA abundance. The term “hungry
codons” has been coined for codons that are awaiting
418
Dr. David W. E. Smith graduated from Swarthmore College
and received his M.D. at Yale, where he also received
training in pathology. He was a Research Associate at the
NIH (1962-1964) with Dr. Bruce Ames, with whom he
worked first on intermediates in histidine biosynthesis and
the histidine operon and later on tRNA in translational
control and on mutagenesis by carcinogenic compounds. He
held appointments at NIH and Indiana University, and since
1969, he has been Professor of Pathology at Northwestern
University Medical School. Most of his research has involved
tRNA function, with some change in emphasis over time
from translational control to the effects of tRNA modifica-
tions on tRNA function. Much of his work was done on
hemoglobin synthesis in reticulocytes and other red blood
cell precursors. He has recently developed an interest in
biological gerontology and is a director of the Buehler Center
on Aging at Northwestern University.
translation by ternary complexes (aminoacyl-tRNA, GTP,
and eF-Tu) with limiting tRNA species (Parker, 1989).
On the other hand, it is argued that the variety and
amounts of proteins synthesized by lower organisms
could not be fine-tuned by tRNA abundance and codon
bias (e.g., Varenne and Lazdunski, 1986; Kurland, 1991).
The overexpression of a single heterologous gene product
in cells without an adapted tRNA content is surely
associated with increased hungry codons. Whatever
translational control is achieved through tRNA content
of homologous systems cannot apply to translation,
resulting in overexpression in heterologous systems. A
few experimental situations have been described in which
there is reduced expression of heterologous genes after
insertion of codons for which there are few cognate tRNA
molecules (e.g., Brinkman et al., 1989; Robinson et al.,
1984; Hoekema et al., 1987; Kane et al., 1992). In one
case, levels of the corresponding mRNA were also re-
duced, suggesting a dependence of mRNA stability on
translation (Hoekema et al., 1987). Expression was
increased in some of these cases and in others after
introduction of genes for scarce tRNA species (e.g.,
Brinkman et al., 1989; Robinson et al., 1984; Rojiani et
al., 1990; Xu and Boske, 1990). Overexpression was
involved in some of these cases (Kane et al., 1992; Xu
and Boske, 1990). In other cases, however, overexpres-
sion of the protein gene product occurred successfully
with the unaltered tRNA content of host cells (e.g., Pak
et al., 1994; Alldread et al., 1992).
Qualitative Effects of tRNA Content on Transla-
tion. Miscoding can be a consequence of tRNA limited
translation. There is an elevated level of miscoding in
overexpression of a heterologous gene for mouse epider-
mal growth factor in Escherichia coli (Scorer et al., 1991).
Generally in translation misincorporation averages 10-4
per site but varies widely from site to site (Parker, 1989).
It is unknown how much misincorporation occurs at
hungry codons.
Biotechnol. Prog., 1996, Vol. 12, No. 4
Figure 1. Diagrammatic representation of tRNA function and
cycling in translation. Aminoacyl-tRNA as a ternary complex
is bound to ribosomes in response to codons in mRNA. Ami-
noacyl-tRNA is first bound to the ribosomal A site and is
translocated to the P site as the amino acid is added to the
elongating polypeptide chain. As the nascent polypeptide chain
is transferred yet again to the next cognate aminoacyl-tRNA,
the previous tRNA leaves the ribosome, to enter a pool first of
free tRNA, then of aminoacyl-tRNA, and finally of ternary
complex, to be recycled. Considerable artistic license has been
taken in this illustration, including the cloverleaf structure for
tRNA and the separation of codons on mRNA. Actually there
is no separation, permitting frameshifting, as described.
The stringent response to amino acid starvation re-
duces misincorporation (Parker, 1989). The stringent
response generally requires elevated levels of uncharged
tRNA, as occurs in amino acid starvation but not in tRNA
limited translation. There is a study, however (Brink-
man et al., 1989), that suggests that translation limited
by tRNA availability may sometimes provoke the strin-
gent response, with a reduction of miscoding. In this
study, tRNA limited translation was associated with
increased levels of ppGpp, the mediator of the stringent
response, although levels of uncharged tRNA were not
elevated.
Premature termination of translation can be another
consequence of low tRNA abundance. In one example,
translation of a foreign gene in an expression system that
lacked a required tRNA species was terminated at the
codon corresponding to the missing tRNA species (Oba
et al., 1991). The mechanism of termination was not
release from the ribosome, as occurs at stop codons, but
termination without release from the ribosome. Prema-
ture termination occurs commonly in protein synthesis,
but it is unknown how much of it occurs at hungry
codons.
Suppression of stop codons depends on a content of
tRNA capable of reading through stop codons with
incorporation of an amino acid (Hatfield et al., 1990). In
ciliates (Cohen et al., 1990), tRNAs that recognize amber
(UAG) and ochre (UAA) codons are aminoacylated with
glutamine, which is incorporated at these codons, and
read-through proteins are synthesized. Ciliate genes
cannot be translated in E. coli, because E. coli tRNA does
not read through amber and ochre codons. A Tetra-
hymena gene for phosphoenolpyruvate mutase was over-
expressed (25% of soluble protein) in E. coli after some
amber codons were changed to glutamine codons and a
gene for a tRNAArg for an arginine codon that rarely
occurs in E. coli was introduced (Seidel et al., 1992).
Synthesis of Mycoplasma and mitochondrial proteins is
also dependent on tRNA that reads through stop codons
(Cohen et al., 1990).
Frameshifting affecting up to 50% of gene products has
been described at hungry codons cognate to scarce tRNA
Biotechnol. Prog., 1996, Vol. 12, No. 4 419

Table 1. Anticodon Loop Base Sequences of Selected tRNA’s Showing Modified Basesa
base position no.
tRNA and source codons read 32 33 34 35 36 37 38
tRNATyr UAU and UAC
B. subtilis C U Q U A A4 or A5 A
E. coli C U Q U A A5 A
yeast C U G F A A4 A
tobacco leaves C U G F A G1 A
human placenta C U Q2 F A G1 A
tRNALys AAA and AAG
B. subtilis N U V5 U U A9 A
E. coli C U U8 U U A7 A
AAA
yeast C U N U U A7 A
rabbit liver C U U9 U U A9 A
AAG
Saccharomyces C U C U U A7 A
rabbit liver C U U9 or C U U A9 or A7 A
tRNAPhe UUU and UUC
B. subtilis C U G3 A A A5 A
E. coli F U G A A A5 A
yeast C3 U G3 A A Y1 A
human placenta and other mammalian cells C3 U G3 A A Y2 A
mouse neuroblastoma C* U G* A A G1 A
a Abbreviations: A4 ) N6-Isopentenyladenosine. A5 ) 2-(Methylthio)-N6-isopentenyladenosine. A7 ) T6A (threonyladenosine). A9

) MS2T6A (((methylthio)threonyl)adenosine). C3 ) 2′-O-Methylcytosine. C* ) Unidentified modified cytosine, not the above. F )

Pseudouridine. G1 ) 1-Methylguanosine. G3 ) 2′-O-Methylguanosine. N ) Unidentified nucleoside. Q ) Queuosine. Q2 )
β-D-Galactosylqueuosine. G* ) Unidentified modified guanosine, not the above. U8 ) 5-((Methylamino)methyl)-2-thiouridine. U9 )
5-((Methoxycarbonyl)methyl)-2-thiouridine. V5 ) 5-(((Carboxymethyl)amino)methyl)-2-thiouridine. Y1 ) Wybutosine. Y2 ) Wybuto-
zoxosine. (Sprinzl et al., 1989. Structures shown in source.)

(Spanjaard et al., 1990; Gallant and Lindsley, 1992;
Kawakami et al., 1993) and aminoacyl-tRNA (Parker,
1989) species. The mechanism of frameshifting with
scarce tRNA seems to be a pause in translation on
ribosomes and slippage of mRNA, as a more readily
translated codon is selected within the adjacent mRNA
base sequence (Pande et al., 1995). In one case frame-
shifting occurred at two tandem codons in the same
reading frame. Translating tandem codons with scarce
tRNA species makes unusual demands, with sequestra-
tion of an already scarce tRNA species in translating the
first codon enhancing its scarcity in translating the
second codon (Varenne and Lazdunski, 1986; Spanjaard
et al., 1990). This is an example of how tRNA content
can affect translation quantitatively in a specific codon
context. The abundance of a tRNA species required for
frameshifting may sometimes orchestrate the multiplicity
of gene products resulting from frameshifting (Kawakami
et al., 1993).
In a related process, an in-frame hop of two codons was
described at a codon cognate to a scarce tRNA species
during overexpression of a gene for bovine placental
lactogen in E. coli (Kane et al., 1992). The hop affected
2% of the placental lactogen, which was missing two
amino acids. The hop did not occur if the codon was
changed to one for which there was more tRNA. Other
examples given in this article of tRNA effects on transla-
tion involve far more than 2% of the protein gene product,
but the above example is included because the mecha-
nism is so clearly related to tRNA scarcity.
Modifications of tRNA Bases
There is a high frequency of modified bases in tRNA
(Bjork, 1992), with modifications complicating the uni-
versality of the genetic code and resulting in chromato-
graphic heterogeneity of tRNA species. tRNA base
modifications are post-transcriptional and enzymatic.
Sometimes there is a mixture of tRNAs modified to
different degrees in the same cell or in different cells of
multicellular organisms. tRNAs of rapidly growing
tumor cells are often hypomodified. Considerable genetic
information is invested in genes for modifying enzymes
in many organisms, suggesting that tRNA modifications
are important (Bjork, 1992).
tRNA modifications are a special case of tRNA content
that is treated separately here. Translation in heterolo-
gous systems uses tRNA with different modifications
than homologous systems. Table 1 shows base sequences
in the anticodon loop (positions 32-38) of tRNALys,
tRNAPhe, and tRNATyr species from mammalian cells and
several microorganisms commonly used as expression
systems (Sprinzl et al., 1989). Modified bases are found
elsewhere in tRNA, but those of the anticodon loop have
the most significant effects on tRNA function in transla-
tion (Bjork, 1992). tRNA modifications can also affect
aminoacylation. The tRNA species shown are used in
the examples described below. Base modifications and
their effects on tRNA function have recently been re-
viewed comprehensively and admirably by Bjork (1992).
Two commonly modified positions in the anticodon loop
are of particular interest.
Position 34. This is the wobble position of the tRNA
anticodon, which pairs with the third base of mRNA
codons. Modifications include small moieties, such as
methyl groups on uridine and also larger moieties such
as Q base (queuosine), which is related to guanine. The
modifications can affect wobble by making the anticodon
more or less rigid (Bjork, 1992).
Position 37. Position 37 is adjacent (3′) to the
anticodon of tRNA. Adenine, guanine, or modified forms
of these bases are in this position in all tRNAs shown in
Table 1 (Sprinzl et al., 1989). Modifying moieties are
commonly bulky, are sometimes called hypermodifica-
tions, and may be hydrophilic (e.g., threonyladenosine)
or hydrophobic (e.g., isopentenyladenosine). Wye base
is a modified form of guanine.
Modifications in position 37 have at least two effects
on translation. (1) They increase base stacking in the
anticodon loop and, therefore, the stability of codon/
anticodon interactions (Bjork, 1992). (2) They occupy
space and thus affect the fit of tRNA into the A and P
sites of ribosomes, which are separated by only 18 Å.
420
There is evidence that selection of tRNA for the A site is
sometimes affected by the tRNA species previously
selected (Smith and Hatfield, 1986).
Qualitative Effects of tRNA Modifications on
Translation. Miscoding, stop codon read-through, and
frameshifting can be affected by tRNA base modifica-
tions, and each results in aberrant proteins that can be
assayed to indicate the efficiency of tRNA in these
processes. Some of the examples described below involve
translation of foreign genes and some of homologous
genes, but in each example, there is a large effect of a
base modification on tRNA function.
An example of miscoding affected by a base modifica-
tion contrasts tRNAPhe with 2-(methylthio)-N6-isopenten-
yladenosine in position 37, which did not incorporate
phenylalanine in response to the leucine codon CUU, and
tRNAPhe that has unmodified adenosine in position 37
(Wilson and Roe, 1989). The unmodified tRNAPhe mis-
incorporated an amount of phenylalanine in response to
CUU that was approximately equal to leucine incorpora-
tion from tRNALeu at CUU sites. The unmodified
tRNAPhe was the product of a foreign tRNAPhe gene that
was overexpressed 11-fold compared to homologous
tRNAPhe. Hypomodification was a consequence of over-
expression. It has been shown (Diaz and Ehrenberg,
1991) that modification of adenine in position 37 affects
codon/anticodon interaction. There is less miscoding with
tRNAPhe with modified adenosine because there are fewer
noncognate codon/anticodon interactions.
An example of stop codon suppression affected by tRNA
base modification and resulting in a large change in
protein synthesis is the synthesis of a high molecular
weight read-through protein from the tobacco mosaic
virus genome, probably a viral replicase subunit, in
tobacco protoplasts and cell-free extracts of tobacco leaves
(Beier et al., 1984; Bienz and Kubli, 1981). Synthesis of
the read-through protein depends on suppression of a
UAG stop codon in a specific codon context (Skuzeski et
al., 1991) by tRNATyr with unmodified guanine in position
34. tRNATyr with Q base in this position, as found in
many other kinds of cells, does not suppress the stop
codon, and the read-through protein is not synthesized.
In tobacco leaves, the read-through protein constitutes
60% of the gene product.
Frameshifting is sometimes affected by tRNA modifi-
cations. Synthesis of the γ subunit of E. coli DNA
polymerase III holoenzyme occurs only if there is frame-
shifting (Tsuchihashi and Brown, 1992) in a mRNA codon
context where there are tandem nonidentical lysine
codons in the sequence A-AAA-AAG. There is 80%
frameshifting in E. coli, in which there is a single tRNALys
species that has 5-((methylamino)methyl)-2-thiouridine
in position 34. It has weak interaction with AAG codons
and stronger interaction with AAA. The single tRNALys
of E. coli frameshifts by -1 in the above context to read
AAA. There is no frameshifting when the same gene is
translated in eukaryotic cells, in which there are two
tRNALys species, one of which has unmodified uridine in
position 34 and has strong interaction with AAG. Frame-
shifting could be an unexpected result in translating a
mammalian gene in an E. coli expression system, with
synthesis of an aberrant protein. Fifty percent frame-
shifting has been observed in translating A-AAA-AAG
of a retroviral gene in E. coli (Weiss et al., 1989). This
frameshifting is not to be confused with the frameshifting
that occurs at the gag/pol gene interface in translating
retrovirus RNA in eukaryotic cells (Hatfield et al., 1990).
Quantitative Effects of Modifications on Transla-
tion Based on tRNA Choice. There are many ex-
amples (e.g., Smith and Hatfield, 1986) of preferential
Biotechnol. Prog., 1996, Vol. 12, No. 4
use in translation of one competing iso-accepting tRNA
species over another based on base modifications. Pref-
erences do not result in aberrant proteins because codons
are read correctly by any of the competing tRNA species,
but tRNA choice can affect translation quantitatively.
The tRNA species preferred at a particular site may not
be the tRNA species that is most abundant in intact cells
or the tRNA species that is generally preferred or that
functions most rapidly in amino acid incorporation. The
result may be slowed elongation of the nascent peptide
and reduced translation of the gene product.
Some preferences are related to effects of modified
bases on codon/anticodon interactions. For example, E.
coli tRNATyr with Q base in position 34 binds twice as
efficiently to ribosomes in the presence of each of the
tyrosine codons, UAU and UAC, as tRNATyr with gua-
nosine in position 34 (Noguchi et al., 1982).
Modifications in position 37 also affect tRNA prefer-
ences. For example, tRNAPhe with guanosine in this
position is preferred in the incorporation of phenylalanine
into most sites of the globin subunits by rabbit reticulo-
cyte lysates, but tRNAPhe with Wye base in position 37
is preferred in the incorporation of a few phenylalanine
residues encoded by each of the phenylalanine codons,
UUU and UUC. Translation of the first UUC in each of
the two tandem UUC codons in mRNA for rabbit β-globin
shows the commonly observed preference for the hypo-
modified tRNAPhe, but translation of the second tandem
UUC codon prefers the tRNAPhe with Wye base in each
case (Smith et al., 1985). This may be an example of
tRNA interaction on ribosomes affecting tRNA choice,
and the choice may limit the synthesis of β-globin and
may have a role in the coordination of R- versus β-globin
synthesis.
Problems and Opportunities
In the recent volume of Methods in Enzymology (Vol-
ume 185) devoted to gene expression technology, David
V. Goeddel (1990), the editor, wrote that “The expression
of foreign proteins is still largely empirical. There is no
set of hard-and-fast rules to follow. In fact, a particular
protein is almost as likely to be an exception as to follow
any set of rules.” The present article does not offer a set
of rules, but instead, it provides reasons why rules may
never be found or why rules may be applicable only at
the level of codon context. At the same time it provides
reasons why tRNA should be of increased concern in
translating proteins from heterologous genes.
In cases of low gene expression, the possibility that
translation is the problem should be considered, and it
should be determined whether mRNA is synthesized but
translated poorly. There is a need for systematic data
about tRNA species contents of expression systems that
can be matched to codon frequencies in genes, so that
mismatches of codon biases and tRNA contents are
avoided. Low translation should be attempted in other
expression systems, and reasons for differences, which
may be related to tRNA, should be investigated.
Protein products of gene expression should be exam-
ined for errors in amino acid sequence which, as exempli-
fied above, can result from premature termination, stop
codon read-through, frame shifting, and hops, as well as
miscoding. Some errors occur at low frequency, but most
of those described above affect most molecules of specific
gene products. Some errors in amino acid sequence have
little effect on the biological properties of proteins, but
others have large effects. Alterations in tryptic peptide
patterns are sometimes sufficient to indicate qualitative
errors in translation (Kane et al., 1992). Products of
Biotechnol. Prog., 1996, Vol. 12, No. 4
translation in which there is read-through or premature
termination can be detected by SDS-PAGE.
Translation of heterologous genes that are overex-
pressed provides excellent opportunities to study tRNA
function at the levels of codon context and tRNA interac-
tions on ribosomes. Concerning translation of mRNA,
heterologous genes can be changed to alter codon bias,
suppressed codons can be inserted, and codon contexts
of interest can be constructed.
It is more difficult to change tRNA in expression
systems. Genes for tRNA species have been introduced
into heterologous cells to change tRNA content (Brink-
man et al., 1989; Hoekema et al., 1987; Rojiani et al.,
1990; Wilson and Roe, 1989), but expression of tRNA
genes in heterologous systems may be different from
homologous cells and must be determined (Bossi and
Ciampi, 1983). It is possible to achieve overexpression
of tRNA species to alter tRNA content (e.g., Wilson and
Roe, 1989; Borel et al., 1993; Pak et al., 1994). It must
be determined that the tRNA is correctly aminoacylated
and that aminoacylation has not become limiting in
protein synthesis. Site-specific mutagenesis of tRNA
genes permits changes in the four canonical bases, but
base modifications are post-transcriptional and depend-
ent on host cell enzymes. Genes for base modifying
enzymes have been isolated and cloned (Dihanich et al.,
1987), but even if they are expressed in heterologous cells,
their usefulness in modifying specific tRNA species will
be variable. Some modification occurs at the tRNA
precursor level, some occurs in the nucleus, and some
requires multiple reactions (Bjork, 1992).
Conclusions
In conclusion, the examples in this article show that
tRNA content and modifications can have large effects
on gene expression through tRNA function in translation.
In translating heterologous genes, the host cell tRNA can
affect the synthesis of gene products both quantitatively
and qualitatively. Translation needs to be of greater
interest in the expression of heterologous genes in
expression systems.
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Accepted August 25, 1995.X
BP950056A
X Abstract published in Advance ACS Abstracts, February 1,
1996.