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Journal of Bacteriology, September 1998, p. 4339-4343, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Efficiency and Frequency of Translational Coupling
between the Bacteriophage T4 Clamp Loader Genes
Michael Y.
Torgov,
Deanna M.
Janzen, and
Michael K.
Reddy*
Chemistry Department, University of
Wisconsin-Milwaukee, Milwaukee, Wisconsin
Received 1 October 1997/Accepted 18 June 1998
 |
ABSTRACT |
The bacteriophage T4 DNA polymerase holoenzyme is composed of the
core polymerase, gene product 43 (gp43), in association with the
"sliding clamp" of the T4 system, gp45. Sliding clamps are the
processivity factors of DNA replication systems. The T4 sliding clamp comes to encircle DNA via the "clamp loader" activity inherent in two other T4 proteins: 44 and 62. These proteins assemble into a pentameric complex with a precise 4:1 stoichiometry of proteins
44 and 62. Previous work established that T4 genes 44 and
62, which are directly adjacent on polycistronic mRNA
molecules, are
to some degreetranslationally coupled. In the present
study, measurement of the levels (monomers/cell) of the clamp
loader subunits during the course of various T4 infections in
different host cell backgrounds was accomplished by quantitative
immunoblotting. The efficiency of translational coupling was obtained
by determining the in vivo levels of gp62 that were synthesized when
its translation was either coupled to or uncoupled from the upstream
translation of gene 44. Levels of gp44 were also measured
to determine the relative stoichiometry of synthesis and the percentage
of gp44 translation that was transmitted across the intercistronic
junction (coupling frequency). The results indicated a coupling
efficiency of ~85% and a coupling frequency of ~25% between the
44-62 gene pair during the course of infection. Thus,
translational coupling is the major factor in maintaining the 4:1
stoichiometry of synthesis of the clamp loader subunits. However,
coupling does not appear to be an absolute requirement for the
synthesis of gp62.
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INTRODUCTION |
Genes 44 and
62 of bacteriophage T4 produce two proteins that are
essential for the replication of the phage DNA (1). These proteins assemble into an oligomeric complex, gp44-gp62 (gp44/62), that
possesses very low intrinsic ATPase activity. The ATPase activity of
the gp44/62 complex is greatly stimulated upon its specific interaction
with another T4-encoded protein, gp45 (14, 23). gp45 is the
"sliding clamp" of the T4 system and is structurally homologous to
the other known sliding clamps, namely, the 
subunit of
Escherichia coli and the eukaryotic proliferating cell
nuclear antigen (20, 21). In vitro studies in a variety of
laboratories have led to the consensus view that the role of gp44/62
during a T4 infection is to chaperone the gp45 protein to DNA (16, 30). Upon binding to DNA, the gp44/62 complex imparts a transient discontinuity to the ring-like structure of the T4 sliding clamp, resulting in the gp45 protein being topologically deposited onto double-stranded DNA (3, 22). That is, the gp44/62
complex is a "clamp loader." Although it is presumed that in
vivo this function is driven by the hydrolysis of ATP, the exact
molecular mechanism by which this event occurs remains unknown. The
role of gp44/62 as a clamp loader is catalytic (16). Once
loaded onto DNA, the gp45 sliding clamp subsequently associates with the T4 DNA polymerase (gp43) to form the processive holoenzyme (9,
17, 31, 34).
The T4 clamp loader is assembled upon the tight binding of one gp62
subunit to a gp44 tetramer (15, 35). The in vivo mechanism by which this strict 4:1 stoichiometry of the gp44/62 complex is
maintained remains unknown. It is to be appreciated that the gp44/62
complex is similar in both sequence and function to the E. coli 
complex and eukaryotic RF-C (19, 28).
Furthermore, the latter two clamp loaders are also composed of five
subunits; however, the implications of the conservation in evolution of this pentameric stoichiometry have yet to be determined.
The 44 and 62 genes occur in a region of the T4
genome clustered with other genes required for DNA replication. The
genes of the T4 replication complex are arranged so that they may be transcribed as a cassette including genes 45, 44,
62, regA, and 43. They are expressed
early in the infection cycle, and the transcripts are polycistronic
(11). Transcription proceeds from early or middle promoters
upstream of gene 45. There are strong consensus translation
initiation regions (TIR) serving genes 45 and 44. The polycistronic message is subject to at least two known forms of
translational regulation. One is repression due to direct binding of
the RegA protein to the TIR of gene 44 (37). The
second form of regulation is translational coupling (see references
6, 13, and 25 for reviews). This
event occurs when translation of a distal gene on a polycistronic mRNA
is strongly, or exclusively, dependent upon translation of a gene
immediately upstream. In 1979, the laboratory of Karam initially
provided evidence that the expression of gene 44 and that of
gene 62 are translationally coupled (18).
However, reflecting an inability to detect the low levels of gp62
produced, the precise degree of translational coupling was not
determined by those investigators. In the current study, by employing
specific antibodies and an extremely sensitive chemiluminescence
detection protocol, we have quantitatively determined the levels of
gp62 present in vivo during T4 infections. We have also examined the
degree of translational coupling as a first step in determining how
strict stoichiometry may be established for the gp44/62 complex.
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MATERIALS AND METHODS |
Strains, media, and growth conditions.
E. coli B
strain Nap IV (sup-1 hsdRK
hsdMK+
hsdSK+ thi)
(26) was used as the suppressing host for T4 amber mutant
44amN82 (gene 44) or 43amB22 (gene
43). sup-1 is a serine-inserting amber (UAG)
suppressor. The wild-type (sup-0) counterpart of the Nap IV
strain was used as the nonsuppressing host for T4 amber mutants, as
well as for infection by wild-type bacteriophage T4 strain D
(T4D+). Cells were grown at 37°C in glycerol Casamino
Acids medium supplemented with thiamine (10 µg/ml) in a rotary shaker
to an A600 of ~0.6, which corresponded to a
cell density of ~1.1 × 109/ml. Standard plate count
assays were performed to determine the exact cell density. The cells
were then infected (multiplicity of infection of 5) with bacteriophage
that had been previously mixed with a small volume of phage diluent
buffer (5) containing L-tryptophan (final
concentration of 5 µg/ml). The number of uninfected cells at 1 min
after phage addition was determined by the standard plate count assay
to be 1% or less. This protocol ensured a nearly simultaneous
infection round and yielded the actual number of infected cells. At
certain time points during the infection round, 0.25-ml aliquots were
withdrawn and phage development was immediately arrested by the
addition of 50 µl of 6× lysis buffer (0.37 M Tris [pH 6.8], 6 mM
dithiothreitol, 6% [wt/vol] sodium dodecyl sulfate [SDS], 30%
[vol/vol] glycerol, 0.01% bromophenol blue). The aliquots were
boiled for 5 min and then stored frozen at 
20°C.
Polyclonal antibody against gp44 and gp62.
Anti-gp62 and
anti-gp44 sera were produced in collaboration with Cynthia Sommer
(Biological Sciences, University of Wisconsin-Milwaukee) and Berri
Forman (Animal Care Facility, University of Wisconsin-Milwaukee) by
following standard protocols. The antibodies were affinity purified
from serum on a protein A column (5-ml cartridge) in accordance with
manufacturer (Bio-Rad) specifications. The resulting antibodies were
tested by immunoblotting against cell lysates and found to have high
specificity for gp62 or gp44, although some cross-reactivity with
E. coli proteins was retained.
Quantitative immunoblotting.
Immunoblotting and detection by
chemiluminescence was employed to quantify the gp44 and gp62 levels in
the lysed time point aliquots. The aliquots were sonicated to shear
DNA, and equal numbers of infected cells (in 6- to 12-µl portions of
the aliquots) were then fractionated via discontinuous
SDS-polyacrylamide gel electrophoresis on a 15% polyacrylamide
minigel. Electrophoresis was performed in 49 mM Tris-384 mM
glycine-0.1% SDS (pH 8.5). Fractionated proteins were electroblotted
in a high-pH transfer buffer [10 mM
3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), pH 11; 10%
methanol] onto a nitrocellulose membrane (Life Technologies, Inc.) by
using a Mini Trans-blot apparatus (Bio-Rad). The blots were blocked
overnight at 4°C with 0.2% (wt/vol) casein and then incubated with
anti-gp62 and/or anti-gp44 antibodies in phosphate-buffered saline. The
blots were developed by using a chemiluminescence system with the
reagents and alkaline phosphatase-conjugated secondary antibody
supplied by the manufacturer (Tropix).
For quantitation of gp44 and gp62 bands, serial dilutions of purified
gp44/62 were included on every gel. Via this method, gp44 and gp62
could be detected at 50 and 10 pg per lane, respectively. Quantitation
of gp62 over a linear range of 20 to 900 pg was achieved by using a
standard dilution-response curve constructed for each immunoblot.
Quantitation of gp44 and gp62 was performed by comparing the
intensities of bands on Hyperfilm ECL (Amersham), using a nonspecific
cross-reactive E. coli protein band as an internal reference
for the amount of lysate loaded per lane. Due to the narrow linear
response range of the film, several exposures were necessary to assign
the correct optical density (OD) value to the low and high ends of the
gp44 and gp62 standards. The photographic negatives of the immunoblots
were scanned on a Color OneScanner (Apple) set to a resolution of 600 dots/in. in the grayscale mode. The scanned images were converted to a
histogram with NIH Image 1.62 software, through which the OD could be
plotted as a response profile. The total OD of a desired band was
obtained by defining a rectangular area around it, summing the OD along
the short axis of the rectangle, and then using the wand tool facility
to measure the peak area in the resulting one-dimensional plot.
Irregular baselines were defined by plotting a section adjacent to the
bands. The areas under the histogram peaks were quantitated. The ratios of the ODs of any two neighboring gp44 or gp62 standards at the low and
high ends of the range at two (or more) exposures were averaged and the
mean value was used to correct the otherwise over- or underexposed
standard at the high or low end, respectively.
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RESULTS AND DISCUSSION |
Synthesis of gp44 and gp62 by 44amN82.
In the course of
T4 infection, a heterogeneous collection of mRNAs containing the coding
sequences for genes 44 and 62 is produced. In all
cases, gene 62 is found directly downstream of gene
44, and translation of gene 62 is known to be
linked to translation of upstream gene 44. Previously,
translation of gene 62 has not been observed in the absence
of complete gene 44 translation. Karam et al. were unable to
directly observe production of gp62 with gene 44 amber
mutants 44amE4408 and 44amN82, although low levels of gene 62 translation were reasoned to occur
(18). Further, gp62 synthesis was not observed when gene
62 was present on an expression vector in the absence of the
initial portion of gene 44 (33). In this study,
we have determined the levels of gp44 and gp62 produced during
infections by 44amN82 under suppressing and nonsuppressing
conditions via quantitative immunoblotting. We have also been able to
demonstrate the synthesis of gp62 in the absence of complete gene
44 translation.
The results of infections of the
E. coli suppressing and
nonsuppressing hosts by
44amN82 are shown in Fig.
1. In the infection
of the
suppressing host (
sup-1), gp44 and gp62 were easily
detectable
by 3.5 min postinfection (p.i.) at levels of 230 and 70 monomers
per cell, respectively. At 5 min p.i., these levels increased
to 1,400 gp44 and 400 gp62 monomers per cell. A final measurement
was
taken at 10 min p.i., when early protein synthesis is nearly
complete. At that point, the level of gp44 was 6,700 monomers
per
cell, while there were 1,600 monomers of gp62 per cell.
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FIG. 1.
Immunoblot analysis for detection and quantitation of
gp44 and gp62 in extracts of gene 44 amber mutant
(44amN82) phage T4-infected cells. Aliquots containing equal
numbers of infected cells were harvested at different times p.i., and
phage development was arrested immediately. Samples of these whole-cell
lysates were fractionated on an SDS-15% polyacrylamide gel and then,
after transfer by electroblotting to a nitrocellulose membrane, probed
with anti-gp44 and -gp62 polyclonal antibodies. Proteins were
visualized with a chemiluminescence detection protocol as described in
Materials and Methods. Serial dilutions of the purified gp44/62 complex
were run on each gel (standards, pg). Arrowheads indicate gp44 and gp62
in the cell lysates at fixed time points (minutes post-infection)
produced during the infection of a suppressing (sup-1)
E. coli host ("coupled") or a nonsuppressing
(sup-0) host ("uncoupled") with 44amN82. To
account for greater gp44 production in the suppressor host
("coupled"), the 10-min lysate was diluted twofold with control
lysate. The control (0 min) was a whole-cell lysate just before phage
addition. A portion of an overexposed blot showing gp62 is shown below
to emphasize the difference in gp62 translation during infection of
suppressor ("coupled") and nonsuppressor ("uncoupled") hosts.
The higher-molecular-weight band is due to cross-reactivity of the
anti-gp62 antibodies with E. coli protein.
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|
A second round of infections was performed by using
44amN82 and the nonsuppressing
E. coli host
(
sup-0). The amber mutation
occurs in the middle
portion of gene
44, producing a truncated
protein which is
about 50% smaller than wild-type gp44 (
40).
The truncated
gp44 was not detected by our assay. Synthesis of
gp62 was detected in
the absence of complete gene
44 translation,
albeit at much
lower levels than when gene
44 translation proceeded
unhindered. At 3.5 min p.i., 15 molecules of gp62 per cell were
detected. At 5 min p.i., there were 100 molecules of gp62 per
cell,
while the level rose to 360 molecules per cell at 10 min
p.i.
Clearly, while gene
44 translation was important for
efficient gene 62 translation, it was not an absolute requirement. The
gene
44 amber mutant has a compromised ability to replicate
DNA
in a nonsuppressing host (
1). Deficiency in DNA
replication
will cause a lower number of T4 templates, which could
decrease
the level of gene
44 and gene
62 encoding mRNAs. This would lead
to a lower level of gp62 produced by
the uncoupled pathway, yielding
an artificially high degree of
translational coupling. However,
the various mRNA species containing
gene
44 and gene
62 are transcribed
from either
early or middle promoters (
11), which are utilized
before
the onset of DNA replication. It can be postulated that
deficiency of
DNA replication should not greatly affect the level
of the gene
44 and
62 messages or the amounts of gp44 and
gp62
produced. To confirm this argument, we carried out infections
of
suppressor and nonsuppressor hosts with
43amB22.
Synthesis of gp44 and gp62 by 43amB22.
Gene
43 codes for the T4 DNA polymerase. The mutation
amB22 causes truncation of the carboxyl-terminal 20% of the
protein. The mutant gp43 possesses DNA binding affinity and exonuclease activity but cannot interact with DNA polymerase accessory proteins and
exhibits no polymerase activity. Thus, in a nonpermissive host,
43amB22 is deficient in DNA replication (27).
Figure
2 depicts the levels of the clamp
loader proteins during infections of
E. coli
suppressor and nonsuppressor hosts with
43amB22. In the
infection of the suppressor host, gp44 and gp62
were detected at 3.5 min p.i. at levels of 600 and 100 monomers
per cell,
respectively. These levels rose to 2,700 gp44 and 800
gp62
monomers per cell at 5 min p.i. At 10 min p.i., there were
7,700 gp44
and 2,700 gp62 monomers present per cell.
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FIG. 2.
Lack of DNA replication does not affect the translation
of gene 44-gene 62 mRNA nor the apparent
stoichiometry of gp44/62 complex synthesis. The DNA
polymerase-deficient T4 mutant (43amB22) produced gp44 and
gp62 during the infection of a sup-0 (nonsuppressor) host in
amounts similar to those of a sup-1 (suppressor) E. coli host, as determined by quantitative immunoblotting (Fig. 1).
The 4:1 stoichiometry was not changed.
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|
Under nonsuppressing conditions, results at 3.5 and 5 min p.i. are
nearly identical, within the range of experimental error,
to those
found under suppressing conditions. For example, at 3.5
min p.i., there
were 420 gp44 and 130 gp62 monomers present per
cell in the
nonsuppressor host while there were 600 and 120 monomers
of gp44 and
gp62, respectively, per cell under suppressing conditions.
At 10 min
p.i., the levels in the nonsuppressor host were only
slightly lower
than those found under suppressing conditions.
The observation that the
levels of gp44 and gp62 produced are
similar in both infections
confirms that DNA replication has little
or no effect on the amounts of
gp44 and gp62 produced, at least
during early portions of T4
infections.
The amounts of gp44 and gp62 produced by
43amB22 are similar
to the levels found in wild-type infections. Burke et al. measured
the
amounts of gp44 and gp62 synthesized between 3.5 and 7 min
p.i. by
using
14C labeling. For infection by wild-type phage,
2,900 gp44 and 700
gp62 molecules per cell were detected during this
time period
(
4). Our results at 5 min from
43amB22 infections, in both
suppressor and nonsuppressor
backgrounds, agree well with these
values. Infections with wild-type
phage yielded 2,500 molecules
of gp62 per cell after 10 min
(results not shown). Our results
at 10 min p.i. with
43amB22
in both backgrounds again correspond
to the wild-type value. It
appears that the levels of gp44 and
gp62 detected in the
43amB22 infections of the suppressor and
nonsuppressor
hosts represent the amounts of these proteins produced
in wild-type
infections.
The levels of both proteins are higher in both
43amB22
infections than in the
44amN82 infection of the suppressor
host (Table
1). When gene
44 is translated from
44amN82 in the
sup-1
background,
there is competition between the suppressor tRNA and
release factors
for the amber codon, leading to less than 100%
suppression efficiency
(
7,
10) and a subsequent
decrease in the amount of gp44 synthesized.
As gene
62 is
translationally coupled to gene
44, the decrease
in the
level of gp44 produced is reflected as a decrease in the
amount of
gp62 synthesized compared to wild-type gp62 levels.
Degree of translational coupling.
Translational coupling
occurs when translation of a downstream gene on a polycistronic mRNA is
dependent upon translation of a gene immediately upstream
(29). Features of the intercistronic region of genes
44 and 62 are illustrated in Fig.
3. Factors which may contribute to the
translational coupling of genes 44 and 62 are the
facts that (i) the start codon of gene 62 is one nucleotide downstream from the stop codon of gene 44 (33),
(ii) the Shine-Dalgarno region from gene 62 is weak by
statistical criteria, and (iii) a putative mRNA secondary structure
could sequester part of the gene 62 TIR (36).
Translation of gene 44 could increase the translation of
gene 62 by unfolding local secondary structure, by
increasing the frequency of favorable ribosome interaction with the
gene 62 TIR via an increase in the local concentration of
ribosomal subunits, or by a combination of these two mechanisms (12, 32).
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FIG. 3.
Schematic of a portion of the polycistronic mRNA
containing coding regions for gp44 and gp62. The expanded portion
illustrates features of the intercistronic region, including the stop
codon for gene 44, the start codon for gene 62,
and the Shine-Dalgarno (SD) region for gene 62. An imperfect
inverted repeat implicated in a putative mRNA secondary structure is
indicated by the opposing arrows (36).
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|
We define the efficiency of translational coupling by comparing the
amounts of gp62 produced in the
44amN82 infection of the
nonsuppressor host (uncoupled translation) to levels produced
in the
43amB22 infections (coupled translation). As the gp62 levels
determined in the
44amN82 infection of the suppressor host
were
reduced by inefficient suppression, they do not represent the
true
amounts of gp62 produced by coupled translation. Furthermore,
the gp62
levels determined from the
43amB22 infections were not
affected by the amber suppression and are, in fact, directly comparable
to wild-type levels (see above). For example, at 3.5 min p.i.,
the
amount of gp62 produced by
44amN82 under nonsuppressing
conditions
was 12.5% of the amount produced by
43amB22 in
the infection of
the suppressing host (Table
1). This indicates an
efficiency
of translational coupling of 87.5%. The overall average
(3.5,
5, and 10 min p.i.) efficiency of translational coupling was 86%
± 3%.
When the amount of uncoupled gp62 synthesis is subtracted from
the overall gp62 amounts, the percentage of gene
44 translation
that is transmitted across the intercistronic
junction can be
calculated (Table
2).
Approximately one-quarter, 24% ± 4%, of
gene
44 translation events lead to reinitiation at the gene
62 TIR.
This coupling frequency appears to be the major factor in
controlling
the stoichiometry of synthesis of the clamp loader
subunits.
An important consideration is the position of the amber mutation in
44amN82. This mutation is known to occur at Gln 171 (
40).
This would result in a truncated gene
44-encoded protein almost
50% smaller than wild-type gp44
(which has 319 amino acids). As
translational coupling is related to
the distance between the
start and stop codons, the large increase in
the distance between
the stop and start codons introduced by the amber
mutation should
abolish translational coupling. That is, termination
will occur
well before the elongating ribosomes reach the
intercistronic
region. In the infections of the nonsuppressing host by
44amN82,
there is no apparent mechanism for melting of the
putative RNA
secondary structure in the gene
44-62 intercistronic region, yet
gp62 is still produced. This suggests that
the positioning of
the terminating ribosome near the gene
62 TIR (increase in local
concentration) is the major contribution to
translational coupling
in this system. Further study is necessary to
separate secondary-structure
contributions from the TIR effect for this
gene pair.
Stoichiometry of gp44 and gp62 synthesis.
The T4 clamp loader
complex maintains a strict 4:1 stoichiometry. The gene 44 and gene 62 messages are present in a one-to-one ratio;
however, the stoichiometry of synthesis observed when all instances of
coupled translation were considered was 3.5 ± 0.7. This gives a
very slight excess of gp62 subunits produced when considering
complex assembly. If the coupling mechanism were not active,
approximately eightfold lower amounts of gp62 would be produced.
This would lead to the production of a vast excess of gp44 monomers. It
appears that this gene pair has evolved such that there is minimal
translation of gene 62 unless the partner gp44 subunits are
expressed, and the frequency of translational coupling aids
stoichiometry of synthesis by allowing one-quarter of all gene
44 translation events to be transmitted across the intercistronic junction.
In the majority of known examples of translational coupling, the
coupling exists as a means of regulation where expression
of the
downstream gene product is maintained in equimolar or greater
amounts
(
2,
8,
29,
38,
39). This clearly is not the
case with T4
genes
44 and
62. However, if f1 and the related
IKe
phage genes V and VII, the downstream gene product is produced
in
amounts much smaller than those of the upstream gene product
(
12,
24). It is possible that other phages have evolved downregulation
across the intercistronic junction of coupled gene pairs as a
means of
avoiding unnecessary gene product accumulation.
| |
ACKNOWLEDGMENTS |
We gratefully thank N. G. Nossal (NIH) for providing us with
the 44amN82 mutant, L. J. Rhea-Krantz (University of
Alberta) for providing the 43amB22 mutant, P. Gauss (Western
State College) for the gift of bacteriophage T4D+ and
E. coli Nap IV sup-0 and Nap IV sup-1,
and C. Sommer (University of Wisconsin-Milwaukee) for invaluable help
with the production of antibodies. M.K.R. thanks Gul Afshan for her
constant support and guidance.
This work was supported by an NSF Early Faculty Career Award to
M.K.R. M.K.R. is a Shaw Scientist (Milwaukee Foundation).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Chemistry
Department, University of Wisconsin-Milwaukee, P.O. Box 413, Milwaukee,
WI 53201-0413. Phone: (414) 229-5355. Fax: (414) 229-5530. E-mail: mkr{at}uwm.edu.
| |
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Journal of Bacteriology, September 1998, p. 4339-4343, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
