BIOCHEMICAL
Vol. 76, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
COUPLING OF PROCESSING OF a-GLUCOSYL TRANSFERASE mRNA WITH TRANSLATION
Kevin
*
Herbert L. Ennis*, Marcia L. Walsh+ R. Lynch+, Janet M. Hill* and Paul S: Cohen+
Roche Institute
of Molecular
Biology,
Nutley,
New Jersey
07110
and + Received
University May
of Rhode
Island,
Kingston,
Rhode
Island
02881
13,1977 SUMMARY
Bacteriophage T4 a-glucosyl transferase mRNA is made as a polycistronic 21s molecule that is processed during normal infection to the commonly found 14.5s species. By using antibiotic inhibitors of protein synthesis, it is possible to distinguish two steps involved in the processing of the 21s polycistronic a-gt mRNA in T4-infected Escherichia coli. There is an initial cleavage to an 18s molecule that does not require protein synthesis. However, the next step, the conversion of the 18s into the 14.5s molecule, requires simultaneous protein synthesis.
Eukaryotic
tRNA,
by nucleolytic
cleavage
In prokaryotic
cells
precursors
that However,
(2,4). cursors.
are
to the there
is
larger
there
The early
rRNA and mRNA are
which
is
dual
messengers
(5).
There
infection processing
transcription, of Escherichia of these
that
that
since coli.
close
no large Inhibition
functional
species
are processed
species
commonly
bacterial
cleaved
a very
which
tRNA and rRNA are
T3 and T7 are
subsequently is
smaller
the mature
mRNAs of phage
molecule,
with
evidence
no evidence
large
cursor
respective
than
is
made as precursors
(l-3).
made as transient found
in the
cell
mRNAs are made as preapparently
made as a single
by RNase III coupling
molecules
to the
of cleavage are
of translation
found
five
indivi-
of the pre-
during
does not
normal affect
mRNAs.
Abbreviations: FA, fusidic A; PM, puromycin; and TET,
acid; CM, chloramphenicol; tetracycline.
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in nny form reserved.
VM, vernamycin
1282 ISSN
0006-291
X
BIOCHEMICAL
Vol. 76, No. 4, 1977
The mRWA that
codes
a-glucosyltransferase
or dextrin
infection,
from T4 DNA is has further
is
located
been
on polysomes
at least
9 ribosomes Since
The 21s molecule to about
required
for
the
molecule
structural
messages
occurs
from
18s to 14.5s
found
cleavages
to the 14.5s
of 23,000
polycistronic.
form
the
probably (see
also
6).
and the 14.5s nucleotides
daltons
are
(9).
required
initiation
fraction
in vivo,
sites
to the nucleotides
mRNA, discrete
containing
found
700-800
with
a minor
2780 nucleotides
subunit
predominantly
on polysomes
mRNA are
about
It
on polysomes
at specific
but only
(7).
The for
the
and termination
The 18s intermediate
sites.
corresponds
1910 nucleotides.
evidence
a-gt
is found
found
mRNA species
to approximately
a-gt
isolated
mRNA made --in vitro
molecule
of a-gt
has in addition
is clearly
We present
a-gt
a-gt),
18s and 21s mRNAs are
sizes
of the a-gt
of the
called
end of a 21s molecule
is
the
1220 nucleotides, synthesis
6-o-
the 21s mRNA is
However,
larger
of approximately
communication,
T4 enzyme,
the a-gt
the 14.5s
few endonucleolytic
portion
to a size
distal
a few discrete
probably
The 21s species
However,
in viva,
corresponds
molecule
14.5s
early
hereafter
and an 18s species,
of the
by a very
(6).
6 ribosomes;
(8).
only
processing
occurs
of the
dextranase,
14.5s
that
containing
14 ribosomes;
rapid
synthesis
at the promoter
shown
at least
--in vivo.
the
RESEARCH COMMUNICATIONS
(EC 2.4.1.2;1,4-a-D-glucan:l,6-+D-glucan
glucosyltransferase 8 min after
for
AND BIOPHYSICAL
which
suggests
in the absence
requires
has similar
that
cleavage
of protein
simultaneous
of the 21s to the
synthesis,
protein
but
synthesis
18s
?-he transition
(Buchanan,
personal
data). RESULTS
During of protein a-gt
synthesis
is
growing
beginning
of experiments on phage
mRNA was inhibited Cells
mRNA
the course
T4 mRNA decay
and that
at 37' were
on the --in vivo we found
an intermediate infected
to be transcribed)
with protein
1283
effects that
of inhibitors processing
18s molecule
was found.
T4, and 4 min later synthesis
of
was inhibited.
(when a-gt RNA
BIOCHEMICAL
Vol. 76, No. 4, 1977
12
4
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6
81012
FRACTION
Fig.
1:
added
6
810
Size of a-gt mKNA made during normal infection-and during infection in the presence of antibiotics. Cells (5 x loo/ml) of E. coli AS19 growing at 37" were infected with wild-type T4D at a multiplicity of 25 as previously described (12). Four min later antibiotic was added at a concentration that rapidly and completely inhibits protein synthesis, and the infection was continued for an additional 5 min (9 min total). (All antibiotics were added to a final concentration of 100 ug/ml, except kasugamycin which was added to a final concentration of 3.4 mg/ml.) No antibiotic was added to the control, which was also infected for a total of 9 min. The cells were rapidly cooled and RNA was isolated as previously described (13). Approximately 100 pg samples of RNA in 0.2 ml of H20 were layered onto 4.8 ml linear 5-20X (w/v) sucrose gradients (0.01 M Tris-HCl, pH 7.5, 0.1 M KCl) and centrifuged in a Spinco SW 50L rotor at 48,000 rpm for 210 min at 2'. Ten 0.5 ml fractions were collected using an ISCO density gradient fractionator connected to a Sargent Welsh SKLG recorder. Each fraction then received 50 ug of E. coli tKNA, 0.14 ml of 1 M sodium acetate , pH 5.2, and 0.72 ml of 95% ethanol. Following overnight precipitation at -15", the samples were centrifuged at 5000 xg for 20 min at 4' and the tubes were inverted and allowed to dry over anhydrous CaS04, for 2 h. Water (0.2 ml) was added to each tube, and the contents were then assayed for a-gt mFUU activities as described previously (12). In control experiments it was found that between 90 and 100% of the RNA in each fraction is recoverable by these methods (data not shown). Heat denaturing the RNA (2 min at 80°, in 1.0 mM sodium acetate, pH 5.2) prior to sucrose gradient centrifugation had no effect on mKNA size and therefore this step was routinely omitted. The media used and conditions for growth and infection have been previously described (12).
was isolated density
4
NUMBER
5 min later
gradient
(9 min after
centrifugation.
to an --in vitro
protein
infection)
and subjected
The RNA in each fraction synthesizing
1284
system
to sucrose
of the
and the resultant
gradient a-gt
was
BlOCHEMlCAl
vol. 76, No. 4, 1977
synthesized
was assayed.
to the relative reflection
Since
concentration
were
phenicol, tetracycline
and vernamycin
hibitors
are
presented
the majority a small
of
T4 infection
at 4 min after
infection,
With
with all
another
involved
in There
study
is
approximately step,
requires sumably
18s.
we are
This
protein
endonucleolytic
(lo),
of the
does not
for
its
(6,8).
portion
at least
that
a broad
peak
the mRNA
to be
as a 14.5s
molecule
(data
shown).
not
that
found
to a species
14.5s
The
molecule, cleavages
apparently are
promoter
observed
are
of
synthesis.
in the are
of
two steps
the 14.5s
mRNA is
the size
known
commonly
protein
These
14.5s.
are
pre-
distal in the
of the cistron.
of processing because
the cleavages
in cells
with
cells
require
a-gt
mRNA accumulated
is
is found
elicitation.
Since
was inhibited
In contrast,
which
to distinguish
although finding,
reduces
18s.
215 molecule
proximal
either
sediments
cleavage
promoter
occur
that
mRNA to its
on the 21s mRNA, (7),
could
to a species
of the 21s a-gt
position
Inhibition
occurs
able
form,
except
that
III,
T4 infection,
some of the mRNA is
of the 18s form into
synthesis
form,
kinase,
antichloram-
to this
a-gt
of approximately
a
of the in-
synthesis
the antibiotic-treated
step
the conversion
protein
cells,
messenger
as in
an initial
to the 14.5s In contrast
in which
deoxynucleotide
the processing
normal
the cleavage
value
as a monocistronic
From this
next
antibiotics,
as well
at 9 min after
to the monocistronic
sedimentation
control
several
present.
is
spiramycin
with
processed
In these
enzyme,
puromycin,
of the polycistronic
21s messenger
early
synthesized
form.
processes
the other
at an average
in the
little
this
clindamycin,
obtained
Clearly,
of cells
chloramphenicol.
the polycistronic
for
1.
at 21s mRNA is also
has been completely
kasugamycin,
mkWA has been
at 9 min after
amicetin,
The results
A.
in Fig.
the a-gt
shoulder
acid,
proportional
The following
fraction.
synthesis:
fusidic
is
in each sample,
in each gradient
protein
RESEARCH COMMUNICATIONS
of enzyme synthesized
of mRNA present
used to stop
erythromycin,
treated
the amount
of the mRNA content
biotics
AND BIOPHYSICAL
of the 18s RNA in protein
synthesis
1285
is
the absence required
for
of protein
synthesis
the formation
of
BIOCHEMICAL
Vol. 76, No. 4, 1977
a T4 DNA-encoded provides
the proper
enzyme
of the a-gt
independent
experiments,
in these
Because
rapid
phenicol,
which
to the already along
idea
present the mRNA.
may be associated
with
that
1) that
some ribosome protein
processing In this
protein
in
synthesis,
the nuclease
We know, is
in-
of chloramphenicol. of chloram-
on and off the
enzyme are made available case
form.
the presence
movement
processing
of chloramphenicol
synthesis
concentration
mRNA is observed
during
14.5s
itself
present
in the presence
all
of this
process
an already
to the
essentially
to allow that
for
mRNA was processed
to 14.5s
is known the
(Fig.
in the presence
cleavage
we favor
travels
cells
the translation
mRNA configuration
It was seen
portion
hibited
Ul),
enzyme or because
ribosome
to function.
a large from
processing
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
responsible
site(s)
the mRNA susceptible
as the ribosome for
processing
the ribosome. ACKNOWLEDGMENT
This work, in part, was supported by grants GB-37942 from The National Science Foundation and AI-11518 from The National Institutes of Allergy and Infectious Diseases, U.S. Public Health Service to P.S.C. We thank the following individuals for their kind gifts of antibiotics: B. Stearns, The Squibb Institute for Medical Research; P. Koetschet and F. Debarre, Rhone-Poulenc; and G.B. Whitfield, Jr., The Upjohn Co. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Burdon, R.H. (1975) in Brookhaven Symposium in Biology, ed. J.J. Dunn, No. 26, 138-153. Nikolaev, N., Birge, C.H., Gotoh, S., Glazier, K. and Schlessinger, D. (1975) in Brookhaven Symposium in Biology, ed. J.J. Dunn, No. 26, 175-193. Perry, R.P., Bard, E., Hames, B.D., Kelley, D.E., and Schibler, U. (1976) in Progress in Nucleic Acid Research, eds. W.E. Cohn and E. Volkin, Academic Press, N.Y., Vol. 19, 275-292. Altman, S., Bothwell, (1975) in Brookhaven A.L.M., and Stark, B.C. Symposium in Biology, ed. J.J. Dunn, No. 26, 12-25. Dunn, J.J. and Studier, R.W. (1975) in Brookhaven Symposium in Biology, ed. J.J. Dunn, No. 26, 267-276. Natale, P.J., Ireland, C., and Buchanan, J.M. (1975) Biochem. Biophys. Res. Commun. 66, 1287-1293. Natale, P.J. and Buchanan, J.M. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 2513-2517. Walsh, M.L., Pennica, D., and Cohen, P.S. (1976) Arch. Biochem. Biophys. 173, 732-738. Sakiyama, S. and Buchanan, J.M. (1973) J. Biol. Chem. 248, 3150-3154. Sakiyama, S. and Buchanan, J.M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 1376-1380. Gurgo, C., Apirion, D. and Schlessinger, D. (1969) J. Mol. Biol. 65, 205-220. Walker, A.C., Walsh, M.L., Pennica, D., Cohen, P.S., and Ennis, H.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1126-1130. Salser, W., Gesteland, R. and Bolle, A. (1967) Nature 215, 588-591.
1286
Vol. 76, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
COUPLING OF PROCESSING OF a-GLUCOSYL TRANSFERASE mRNA WITH TRANSLATION
Kevin
*
Herbert L. Ennis*, Marcia L. Walsh+ R. Lynch+, Janet M. Hill* and Paul S: Cohen+
Roche Institute
of Molecular
Biology,
Nutley,
New Jersey
07110
and + Received
University May
of Rhode
Island,
Kingston,
Rhode
Island
02881
13,1977 SUMMARY
Bacteriophage T4 a-glucosyl transferase mRNA is made as a polycistronic 21s molecule that is processed during normal infection to the commonly found 14.5s species. By using antibiotic inhibitors of protein synthesis, it is possible to distinguish two steps involved in the processing of the 21s polycistronic a-gt mRNA in T4-infected Escherichia coli. There is an initial cleavage to an 18s molecule that does not require protein synthesis. However, the next step, the conversion of the 18s into the 14.5s molecule, requires simultaneous protein synthesis.
Eukaryotic
tRNA,
by nucleolytic
cleavage
In prokaryotic
cells
precursors
that However,
(2,4). cursors.
are
to the there
is
larger
there
The early
rRNA and mRNA are
which
is
dual
messengers
(5).
There
infection processing
transcription, of Escherichia of these
that
that
since coli.
close
no large Inhibition
functional
species
are processed
species
commonly
bacterial
cleaved
a very
which
tRNA and rRNA are
T3 and T7 are
subsequently is
smaller
the mature
mRNAs of phage
molecule,
with
evidence
no evidence
large
cursor
respective
than
is
made as precursors
(l-3).
made as transient found
in the
cell
mRNAs are made as preapparently
made as a single
by RNase III coupling
molecules
to the
of cleavage are
of translation
found
five
indivi-
of the pre-
during
does not
normal affect
mRNAs.
Abbreviations: FA, fusidic A; PM, puromycin; and TET,
acid; CM, chloramphenicol; tetracycline.
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in nny form reserved.
VM, vernamycin
1282 ISSN
0006-291
X
BIOCHEMICAL
Vol. 76, No. 4, 1977
The mRWA that
codes
a-glucosyltransferase
or dextrin
infection,
from T4 DNA is has further
is
located
been
on polysomes
at least
9 ribosomes Since
The 21s molecule to about
required
for
the
molecule
structural
messages
occurs
from
18s to 14.5s
found
cleavages
to the 14.5s
of 23,000
polycistronic.
form
the
probably (see
also
6).
and the 14.5s nucleotides
daltons
are
(9).
required
initiation
fraction
in vivo,
sites
to the nucleotides
mRNA, discrete
containing
found
700-800
with
a minor
2780 nucleotides
subunit
predominantly
on polysomes
mRNA are
about
It
on polysomes
at specific
but only
(7).
The for
the
and termination
The 18s intermediate
sites.
corresponds
1910 nucleotides.
evidence
a-gt
is found
found
mRNA species
to approximately
a-gt
isolated
mRNA made --in vitro
molecule
of a-gt
has in addition
is clearly
We present
a-gt
a-gt),
18s and 21s mRNAs are
sizes
of the a-gt
of the
called
end of a 21s molecule
is
the
1220 nucleotides, synthesis
6-o-
the 21s mRNA is
However,
larger
of approximately
communication,
T4 enzyme,
the a-gt
the 14.5s
few endonucleolytic
portion
to a size
distal
a few discrete
probably
The 21s species
However,
in viva,
corresponds
molecule
14.5s
early
hereafter
and an 18s species,
of the
by a very
(6).
6 ribosomes;
(8).
only
processing
occurs
of the
dextranase,
14.5s
that
containing
14 ribosomes;
rapid
synthesis
at the promoter
shown
at least
--in vivo.
the
RESEARCH COMMUNICATIONS
(EC 2.4.1.2;1,4-a-D-glucan:l,6-+D-glucan
glucosyltransferase 8 min after
for
AND BIOPHYSICAL
which
suggests
in the absence
requires
has similar
that
cleavage
of protein
simultaneous
of the 21s to the
synthesis,
protein
but
synthesis
18s
?-he transition
(Buchanan,
personal
data). RESULTS
During of protein a-gt
synthesis
is
growing
beginning
of experiments on phage
mRNA was inhibited Cells
mRNA
the course
T4 mRNA decay
and that
at 37' were
on the --in vivo we found
an intermediate infected
to be transcribed)
with protein
1283
effects that
of inhibitors processing
18s molecule
was found.
T4, and 4 min later synthesis
of
was inhibited.
(when a-gt RNA
BIOCHEMICAL
Vol. 76, No. 4, 1977
12
4
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6
81012
FRACTION
Fig.
1:
added
6
810
Size of a-gt mKNA made during normal infection-and during infection in the presence of antibiotics. Cells (5 x loo/ml) of E. coli AS19 growing at 37" were infected with wild-type T4D at a multiplicity of 25 as previously described (12). Four min later antibiotic was added at a concentration that rapidly and completely inhibits protein synthesis, and the infection was continued for an additional 5 min (9 min total). (All antibiotics were added to a final concentration of 100 ug/ml, except kasugamycin which was added to a final concentration of 3.4 mg/ml.) No antibiotic was added to the control, which was also infected for a total of 9 min. The cells were rapidly cooled and RNA was isolated as previously described (13). Approximately 100 pg samples of RNA in 0.2 ml of H20 were layered onto 4.8 ml linear 5-20X (w/v) sucrose gradients (0.01 M Tris-HCl, pH 7.5, 0.1 M KCl) and centrifuged in a Spinco SW 50L rotor at 48,000 rpm for 210 min at 2'. Ten 0.5 ml fractions were collected using an ISCO density gradient fractionator connected to a Sargent Welsh SKLG recorder. Each fraction then received 50 ug of E. coli tKNA, 0.14 ml of 1 M sodium acetate , pH 5.2, and 0.72 ml of 95% ethanol. Following overnight precipitation at -15", the samples were centrifuged at 5000 xg for 20 min at 4' and the tubes were inverted and allowed to dry over anhydrous CaS04, for 2 h. Water (0.2 ml) was added to each tube, and the contents were then assayed for a-gt mFUU activities as described previously (12). In control experiments it was found that between 90 and 100% of the RNA in each fraction is recoverable by these methods (data not shown). Heat denaturing the RNA (2 min at 80°, in 1.0 mM sodium acetate, pH 5.2) prior to sucrose gradient centrifugation had no effect on mKNA size and therefore this step was routinely omitted. The media used and conditions for growth and infection have been previously described (12).
was isolated density
4
NUMBER
5 min later
gradient
(9 min after
centrifugation.
to an --in vitro
protein
infection)
and subjected
The RNA in each fraction synthesizing
1284
system
to sucrose
of the
and the resultant
gradient a-gt
was
BlOCHEMlCAl
vol. 76, No. 4, 1977
synthesized
was assayed.
to the relative reflection
Since
concentration
were
phenicol, tetracycline
and vernamycin
hibitors
are
presented
the majority a small
of
T4 infection
at 4 min after
infection,
With
with all
another
involved
in There
study
is
approximately step,
requires sumably
18s.
we are
This
protein
endonucleolytic
(lo),
of the
does not
for
its
(6,8).
portion
at least
that
a broad
peak
the mRNA
to be
as a 14.5s
molecule
(data
shown).
not
that
found
to a species
14.5s
The
molecule, cleavages
apparently are
promoter
observed
are
of
synthesis.
in the are
of
two steps
the 14.5s
mRNA is
the size
known
commonly
protein
These
14.5s.
are
pre-
distal in the
of the cistron.
of processing because
the cleavages
in cells
with
cells
require
a-gt
mRNA accumulated
is
is found
elicitation.
Since
was inhibited
In contrast,
which
to distinguish
although finding,
reduces
18s.
215 molecule
proximal
either
sediments
cleavage
promoter
occur
that
mRNA to its
on the 21s mRNA, (7),
could
to a species
of the 21s a-gt
position
Inhibition
occurs
able
form,
except
that
III,
T4 infection,
some of the mRNA is
of the 18s form into
synthesis
form,
kinase,
antichloram-
to this
a-gt
of approximately
a
of the in-
synthesis
the antibiotic-treated
step
the conversion
protein
cells,
messenger
as in
an initial
to the 14.5s In contrast
in which
deoxynucleotide
the processing
normal
the cleavage
value
as a monocistronic
From this
next
antibiotics,
as well
at 9 min after
to the monocistronic
sedimentation
control
several
present.
is
spiramycin
with
processed
In these
enzyme,
puromycin,
of the polycistronic
21s messenger
early
synthesized
form.
processes
the other
at an average
in the
little
this
clindamycin,
obtained
Clearly,
of cells
chloramphenicol.
the polycistronic
for
1.
at 21s mRNA is also
has been completely
kasugamycin,
mkWA has been
at 9 min after
amicetin,
The results
A.
in Fig.
the a-gt
shoulder
acid,
proportional
The following
fraction.
synthesis:
fusidic
is
in each sample,
in each gradient
protein
RESEARCH COMMUNICATIONS
of enzyme synthesized
of mRNA present
used to stop
erythromycin,
treated
the amount
of the mRNA content
biotics
AND BIOPHYSICAL
of the 18s RNA in protein
synthesis
1285
is
the absence required
for
of protein
synthesis
the formation
of
BIOCHEMICAL
Vol. 76, No. 4, 1977
a T4 DNA-encoded provides
the proper
enzyme
of the a-gt
independent
experiments,
in these
Because
rapid
phenicol,
which
to the already along
idea
present the mRNA.
may be associated
with
that
1) that
some ribosome protein
processing In this
protein
in
synthesis,
the nuclease
We know, is
in-
of chloramphenicol. of chloram-
on and off the
enzyme are made available case
form.
the presence
movement
processing
of chloramphenicol
synthesis
concentration
mRNA is observed
during
14.5s
itself
present
in the presence
all
of this
process
an already
to the
essentially
to allow that
for
mRNA was processed
to 14.5s
is known the
(Fig.
in the presence
cleavage
we favor
travels
cells
the translation
mRNA configuration
It was seen
portion
hibited
Ul),
enzyme or because
ribosome
to function.
a large from
processing
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
responsible
site(s)
the mRNA susceptible
as the ribosome for
processing
the ribosome. ACKNOWLEDGMENT
This work, in part, was supported by grants GB-37942 from The National Science Foundation and AI-11518 from The National Institutes of Allergy and Infectious Diseases, U.S. Public Health Service to P.S.C. We thank the following individuals for their kind gifts of antibiotics: B. Stearns, The Squibb Institute for Medical Research; P. Koetschet and F. Debarre, Rhone-Poulenc; and G.B. Whitfield, Jr., The Upjohn Co. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
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