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Early in vitro and in vivo transcription of T4 DNA
106
BIOCHIMICAET BIOPHYSICAACTA
BBA 96486
E A R L Y I N V I T R O AND I N V I V O T R A N S C R I P T I O N OF T4 DNA II. T H E F I D E L I T Y OF I N V I T R O T R A N S C R I P T I O N A. J. E. COLVILL Research School o/Biological Sciences, Australian National University, Canberra, A.C.T. (Australia)
(Received October 27th, 1969)
SUMMARY I. The nature of in vitro RNA synthesized during 20 rain at temperatures between 25 and 420 has been examined both as to its physical nature and its content of immediate-early and delayed-early sequences. 2. The results show that there are two classes of RNA transcribed in vitro during this time, which differ in their physical size. The set of smaller molecules which is synthesized very early in vitro and is the precursor for the larger RNA corresponds to in vivo immediate-early RNA. The other set contains sequences in common with both immediate-early and delayed-early RNA. 3- The data shows that this latter set of molecules are composed of co-polymers of immediate-early and delayed-early RNA molecules. It is suggested that some termination factor is missing in the in vitro system.
INTRODUCTION In the previous paper I° the presence of two physically distinct classes of messenger R N A m a d e early after T4 infection was reported. O n the basis of hybridization competition data it was suggested that these classes correspond to the two broad temporal classes observed previously to occur early in infection. The presence of ribosomal and soluble R N A m a d e it difficult to distinguish clearly the in vivo messengers and so it was decided to repeat these experiments using R N A synthesized in vitro.
Previous investigations have indicated that in vitro transcription of the mature T 4 genome closely resemble in vivo transcription. Not only was in vitro transcription asymmetric 1-5 but the RNA appeared on the basis of hybridization competition experiments to be identical with 'early' (or pre DNA replication) RNA 6. This gave rise to the suggestion that the DNA and polymerase themselves were sufficient to ensure fidelity of selection and this fidelity was established possibly at the initiation step. Since this time it has been shown that 'early' protein synthesis in T4-infected Escherichia coli is itself subdivided into several sequences 7. A similar result has also been obtained for RNA synthesis in vivo TM. It has been shown by hybridization competition analysis that during the first 5 rain of infection at 3 °o and the first 12 min at 25 °, the number and relative abundance of different RNA sequences changes. Biochim. Biophys. Acta, 2o9 (197o) lO6-111
THE F I D E L I T Y OF TRANSCRIPTION
in vitro
lO 7
Annealing-competition results of MILANESI et al. 9 showed t h a t in vitro RNA synthesis appeared to parallel the in vivo synthesis in that the RNA synthesized in vitro for only a short time is predominantly restricted to those sequences synthesized in vivo at the very beginning of infection. The question arises as to just what extent this fidelity exists. If early synthesis depends on protein synthesis as is suggested b y the results in the preceding paper ~° then it should not be possible to make delayed-early RNA in vitro, only immediate-early which would be contrary to previous findings e. Therefore the nature of in vitro RNA synthesized over a long period at rapid rates has been examined both as to its physical nature and its content of immediate-early and delayed-early sequences. MATERIALS AND METHODS
Phage infection, RNA preparation for hybridization, gel electrophoresis, and R N A - D N A hybridization were all carried out as described previously 1°. T 4 DNA preparation and in vitro RNA preparation using a DEAE-cellulose fraction of E. coli RNA polymerase n were carried out as described elsewherO ,6. For experiments involving the use of limiting enzyme 0. 4 unit of enzyme per #g of DNA was added to the incorporation mixture. 12.8 units/#g of DNA were added for those experiments performed with excess enzyme. These ratios were determined b y measuring incorporation with various amounts of enzyme and a fixed concentration of DNA. The excess values correspond to the plateau region of this curve and the limiting to the linear portion. One unit of enzyme is defined as incorporating I nmole of labelled nucleoside triphosphate into trichloroacetic acid-precipitable material in I h (ref. I I ) . For electrophoresis of in vitro RNA an aliquot of the incubation mixture was made 0.25 % in sodium dodecyl sulphate and 0.2 M in sucrose 12. The resulting solution was then layered on top of a 2.6 % acrylamide gel and a current of 5 mA per gel applied. It was found that the protein did not cause restriction of the gel as described b y LOENING~a, either due to its low concentration or the presence of the sodium dodecyl sulphate. The gels were washed for 6 h at o ° in two changes of 5 % trichloroacetic acid to remove labelled triphosphates and then sliced into o.5-mm discs and two discs placed on a gummed label. The samples, when dry, were then stuck on aluminium planchets and counted on a Nuclear Chicago gas flow planchet counter. RNA was extracted from the gels for hybridization b y sterilely cutting out the required section from several gels and replacing them in perspex tubes. In all steps of this process great care was taken to avoid ribonuclease contamination. The RNA was then back-electrophoresed into 2 ml of electrophoresis buffer (less sodium dodecyl sulphate) contained in a dialysis bag tied over the end of the tube, the bag dipped into the lower buffer vessel thus making electrical contact. The RNA samples so obtained were bulked, made 0.5 M in NaC1 to precipitate any sodium dodecyl sulphate present and the RNA precipitated from the supernatant with 2.5 volumes of ethanol. The RNA was then dissolved in water and stored frozen. In this paper the two classes of pre-replicative early RNA will be referred to as immediate-early and delayed-early as suggested b y R. H. Epstein. The distinction between these two classes being that in vivo the synthesis of the latter class is dependent on protein synthesis. Biochim. Biophys. Acta, 2o9 (197 o) lO6-111
IO8
A . J . E . COLVILL
R E S U L T S
Fig. I shows the d i s t r i b u t i o n of label in samples t a k e n at various times from an in vitro incorporation system using limiting e n z y m e at 42° a n d then subjected to acrylamide-gel electrophoresis. After I m i n of incorporation there is a broad bim o d a l d i s t r i b u t i o n of low-molecular-weight RNA. The m a x i m u m size of the R N A increases with time a n d s i m u l t a n e o u s l y there is a shift in the t o t a l distribution. Thus those species which migrate further t h a n 4.o cm are not present in samples t a k e n after 2 min. The largest of these R N A molecules, which for convenience have been referred to as Peak I, has a molecular weight of 4.5' lO5 calculated from the d a t a of LOENING14 using R I 7 (mol. wt. I . I . I06) ts as a s t a n d a r d . At 2 m i n the m a x i m u m counts are found 2.o cm from the origin b u t some counts still r e m a i n in the P e a k - I region. As can be seen from Fig. I the position of the m a x i m a increases u n t i l at 20 m i n it is found at the origin.
(--
a(
i
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'
d
64 4~
)o
2o
3o
~o
so
60
70
80
io
20
30
#o
so
60
70
80
Distance moved (cm)
Fig. i. Separation of i~ vitro synthesized RNA by electrophoresis on acrylalnide gels. o.o8-ml aliquots of RNA were taken from the incubation mixture at various times: a, i min; b, 5 rain; c, IO nlin; d, 20 rain.
The disappearance of Peak I from the gel p a t t e r n with time suggested t h a t the species in this region were possibly the precursors for the larger species f o u n d between o a n d 4.o cm (referred to as P e a k II). A pulse-chase e x p e r i m e n t was therefore performed to investigate this possibility. Fig. 2 shows the p a t t e r n o b t a i n e d after a 2Biochim. Biophys. Acta, 209 (197o) lO6-111
lO9
THE FIDELITY OF TRANSCRIPTION i n vilro
Cl
o .c
E
U
10
20
30
40
50
60
70
80
10
20
30
40
5.0
60
70
8.0
Distonce moved (cm~
Fig. 2. Electrophoretic separation of pulse-chased RNA. o.o8-ml aliquots of in vitro R N A w e r e t a k e n from the incubation m i x t u r e s at 20 miD. a. Pulse-labelled w i t h 0.2/~mole/ml ~14C]ATP for 2 mid and t h e n chased w i t h 2 0 / , m o l e s / m l unlabelled A T P to m i n u t e 20 at 3 o°. b. Labelled cont i n u o u s l y for 20 mid at 3 o°.
mid pulse at 3 °0 followed by a cold chase for 18 miD. A 2D-miD incorporation at this temperature is also shown for comparison. The experiment was performed at 3 °0 in order to minimize the incorporation of label into Peak II. The RNA in the two peaks was then examined by hybridization-competition experiments to investigate their correspondence with in vivo RNA species. Labelled RNA corresponding to each of the two peaks was therefore extracted from the gels as described in MATERIALSAND METHODS, Peak I from the I-miD gel and Peak I I from the 2o-min gel. These were then competed with unlabelled in vivo RNA, isolated 2 and 12 miD after phage infection at 25 °. Fig. 3 shows that 9° % of the RNA isolated from Peak I is competed for by RNA extracted 2 min after infection. RNA extracted 12 miD after infection competes for 80 % of this RNA and for 9° % of
~o • t~ E
6o-
~0 ¸ ._E 20. U
,'0
2'0
3;°
,'0
~;0
6'0
70
Unlabelled RNA ( m g / m l )
Fig. 3. Correspondence between in vivo a n d in vitro RNA_: H y b r i d i z a t i o n competition. Competition against R N A labelled with [14C]ATP from Peaks I and I I w i t h various in vivo IZNA's. T h e hybridizations were p e r f o r m e d in the presence of 3o # g / m l T 4 DNA. H y b r i d i z a t i o n in t h e absence of unlabelled RI~A is 8o. 5 % of the i n p u t trichloroacetic acid-precipitable radioactivity for Peak I (ioo % represents 129 counts/miD) and 78.5 % for Peak I I (lOO % represents 1126 counts/miD). O , R N A from Peak I and unlabelled IRNA extracted 12 miD after infection; 0 , R N A from Peak I and unlabelled R N A extracted 2 mid after infection; 71, R N A from Peak I I and unlabelled R N A extracted 12 mid after infection; II, R N A from Peak I I and unlabelled R N A extracted 2 mid after infection.
Biochim. Biophys. Acta, 209 (197 o) i o 6 - i i i
II0
A. J . E. C O L V I L L
P e a k - I I RNA. The RNA extracted 2 min after infection only competes for approx. 15 % of the P e a k - I I RNA. The size of the larger RNA molecules synthesized in vitro in 20 rain at various temperatures with limiting enzyme increases with temperature. This can be seen from Table I which tabulates the results obtained on running these RNA samples on acrylamide gels. If the synthesis is carried out at 42o using a saturating amount of enzyme then the RNA in Peak I I migrates I.O cm which is similar to the mobility observed at 35 °. Rifampicin added 15 sec after the start of the incubation at 42°, using limiting enzyme, did not alter the size distribution of the RNA synthesized after 20 rain. TABLE
I
MIGRATION OF P E A K
I[
RNA
SYNTHESIZED
in vitro
FOR 20 MIN AT V A R I O U S T E M P E R A T U R E S
Temperature o/ synthesis (°)
Distance moved (cm)
25 3° 35 42
2.8 2.1 I.O ~ 0. I
DISCUSSION
The results of the preceding paper 1° demonstrate the synthesis of two classes of RNA during the 'early' phase of T4 infection which differ in their molecular size. T h a t equivalent to immediate-early being of molecular weight less than o.7I. lO 6 and the delayed-early being larger than this. The results presented here show that both of these classes of RNA are synthesized in vitro, however the low-molecularweight species is a precursor for the larger species. This can be seen from the disappearance of Peak I after 2 min of synthesis at 42°, as shown in Fig. I, together with the fact that these counts can be chased into the larger species (Fig. 2) and that once synthesis has commenced there are no new initiations as shown by the addition of rifampicin. The results of Table I show that as nett synthesis increases so the size of the RNA increases. This however does not hold if the synthesis is carried out in the presence of excess enzyme when the average size decreases, this is possibly due to nonspecific binding of polymerase under these conditions. Therefore although at first glance the pattern of in vitro messenger RNA synthesis appears to be similar to that observed for in vivo messenger synthesis there appear to be some basic differences. Firstly the synthesis in vivo of the larger delayedearly is dependent on protein synthesis s,l°, except perhaps for some escape synthesis which occurs after long exposures to chloramphenicol. On this basis the appearance of the larger presumably delayed-early RNA in vitro is unexpected, although this result is in agreement with previous hybridization-competition data 6,1~. Secondly the smaller RNA species synthesized in vitro appear to be precursors to the larger species. The results of Fig. 3 suggest that from the point of view of sequence specificity there is again a discrepancy between in vivo and in vitro messenger RNA. It would Biochim. Biophys. Acta,
209 (197 o) l O 6 - 1 1 1
THE FIDELITY OF TRANSCRIPTION
in vitro
III
appear that the sequences transcribed during the immediate-early phase of infection are essentially the same as those transcribed rapidly in vitro, Both delayed-early and immediate-early in vivo RNA compete almost completely for Peak-I RNA indicating that in the first minute of in vitro synthesis at 42o transcription is restricted to those sequences synthesized in vivo at the very beginning of infection. Delayed-early RNA, RNA extracted 12 rain after infection, competes for most of the RNA in Peak I I however immediate-early in vivo RNA also competes for about 15 % of this RNA. This indicates that somewhere in the order of 15 % of the in vitro P e a k - I I RNA is transcribed from sequences common to the in vivo immediate-early RNA. Approx. 75 °o is transcribed from sequences whose transcription in vivo is dependent on protein synthesis and as shown previously is not commenced until 6 rain after phage infection at 25 ° . Thus although in vitro, as in vivo, there appears to be two physically distinct classes of RNA synthesized with a temporal separation, the similarity is only superficial. I n vitro as in vivo, the immediate-early cistrons are transcribed first and the product of this synthesis is much smaller than that resulting from delayed-early transcription. The larger species synthesized in vitro however are transcribed from both immediate-early and delayed early cistrons, and all immediate-early messenger appears to end up in copolymers of immediate and delayed-early RNA, This means that the mechanism for terminating the immediate-early messengers in vivo is missing in the in vitro system. More interestingly this result also implies that the cistrons coding for immediate and delayed-early messenger must be contiguous on the T 4 genomc. ACKNOWLEDGEMENTS
I would like to thank Dr. D. G. Catcheside in whose Department this work was carried out, along with Drs. D. J. Bennett, E. H. Creaser and E. P. Geiduschek for helpful discussions and comments on this manuscript. The technical assistance ot Miss M. Colley and Mrs. J. Picker is also gratefully acknowledged. REFERENCES I ]V[. HAYASHI, M. N. }-IA.YASHI2kND S. SPIEGELMAN, Proc. Natl. Acid. Sci. U.S., 50 (1963) 664. 2 M. HAYASHI, M. N. HAYASHI AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 51 (1964) 351. 3 E. P. GEIDUSCHEK, G. P, TOCCHINI-VALENTINI AND IV[. SA.RNAT, Proc. Natl. Acad. Sci. U.S., 52 (1964) 486. 4 A. J. E. COLVILL, L. C. KANNER, G. P, TOCCHINI-VALENTINI, M. SARNAT AND E. P. GEIDUSCHEK, Proc. Natl. Acid. Sci. U.S., 53 (1965) 114o. 5 E. P. GEIDUSCHEK, Bull. Soc. Chim. Biol., 47 (I968) 1571. 6 E. P. GEIDUSCHEK, L. SNYDER, A. J. E. COLVILL AND M. SARNAT, J. Mol. Biol., 19 (1966) 541. 7 C. LEVINTHAL, J. HOSODA AND D. SHUE, in J. S. COLTER AND W. PARANCHYCH, Mo~eculat Biology o/ Viruses, Academic Press, N e w York, 1967, p. 71. 8 W. SALSER, A. BOYLE AND R. EPSTEIN, J. Mol. Biol., (197o) in the press. 9 G. MILANESI, E. N. BRODY AND E. P. GEIDUSCHEK, Nature, 221 (1967) lOI 4. io A. J. E. COLVILL, Biochim. Biophys. Acta, 209 (197o) 97. I I M. CHAMBERLIN AND P. BERG, Proc. Natl. Acid. Sci. U.S., 48 (1962) 81. 12 N. R. PACE, D. I-I. L. BISHOP AND S. SPIEGELMAN, Proc. Natl. Acid. Sci. U.S., 58 (1967) 711. 13 U. E. LOENING, Biochem. ]., lO2 (1967) 251. 14 U. E. LOENING, Biochem. J., 113 (1969) 131. 15 }~. F. GESTLAND AND H. BOEDTKER, J. Mol. Biol., 8 (1964) 496. 16 E. P. GEIDUSCHEK, E. ~ . BRODY AND D. L. WILSON, in ]3. PULMAN, Molecular Associations in Biology, Academic Press, N e w York, 1968, p. 163.
Biochirn. Biophys. Acta, 2o9 (197 o) lO6-111
BIOCHIMICAET BIOPHYSICAACTA
BBA 96486
E A R L Y I N V I T R O AND I N V I V O T R A N S C R I P T I O N OF T4 DNA II. T H E F I D E L I T Y OF I N V I T R O T R A N S C R I P T I O N A. J. E. COLVILL Research School o/Biological Sciences, Australian National University, Canberra, A.C.T. (Australia)
(Received October 27th, 1969)
SUMMARY I. The nature of in vitro RNA synthesized during 20 rain at temperatures between 25 and 420 has been examined both as to its physical nature and its content of immediate-early and delayed-early sequences. 2. The results show that there are two classes of RNA transcribed in vitro during this time, which differ in their physical size. The set of smaller molecules which is synthesized very early in vitro and is the precursor for the larger RNA corresponds to in vivo immediate-early RNA. The other set contains sequences in common with both immediate-early and delayed-early RNA. 3- The data shows that this latter set of molecules are composed of co-polymers of immediate-early and delayed-early RNA molecules. It is suggested that some termination factor is missing in the in vitro system.
INTRODUCTION In the previous paper I° the presence of two physically distinct classes of messenger R N A m a d e early after T4 infection was reported. O n the basis of hybridization competition data it was suggested that these classes correspond to the two broad temporal classes observed previously to occur early in infection. The presence of ribosomal and soluble R N A m a d e it difficult to distinguish clearly the in vivo messengers and so it was decided to repeat these experiments using R N A synthesized in vitro.
Previous investigations have indicated that in vitro transcription of the mature T 4 genome closely resemble in vivo transcription. Not only was in vitro transcription asymmetric 1-5 but the RNA appeared on the basis of hybridization competition experiments to be identical with 'early' (or pre DNA replication) RNA 6. This gave rise to the suggestion that the DNA and polymerase themselves were sufficient to ensure fidelity of selection and this fidelity was established possibly at the initiation step. Since this time it has been shown that 'early' protein synthesis in T4-infected Escherichia coli is itself subdivided into several sequences 7. A similar result has also been obtained for RNA synthesis in vivo TM. It has been shown by hybridization competition analysis that during the first 5 rain of infection at 3 °o and the first 12 min at 25 °, the number and relative abundance of different RNA sequences changes. Biochim. Biophys. Acta, 2o9 (197o) lO6-111
THE F I D E L I T Y OF TRANSCRIPTION
in vitro
lO 7
Annealing-competition results of MILANESI et al. 9 showed t h a t in vitro RNA synthesis appeared to parallel the in vivo synthesis in that the RNA synthesized in vitro for only a short time is predominantly restricted to those sequences synthesized in vivo at the very beginning of infection. The question arises as to just what extent this fidelity exists. If early synthesis depends on protein synthesis as is suggested b y the results in the preceding paper ~° then it should not be possible to make delayed-early RNA in vitro, only immediate-early which would be contrary to previous findings e. Therefore the nature of in vitro RNA synthesized over a long period at rapid rates has been examined both as to its physical nature and its content of immediate-early and delayed-early sequences. MATERIALS AND METHODS
Phage infection, RNA preparation for hybridization, gel electrophoresis, and R N A - D N A hybridization were all carried out as described previously 1°. T 4 DNA preparation and in vitro RNA preparation using a DEAE-cellulose fraction of E. coli RNA polymerase n were carried out as described elsewherO ,6. For experiments involving the use of limiting enzyme 0. 4 unit of enzyme per #g of DNA was added to the incorporation mixture. 12.8 units/#g of DNA were added for those experiments performed with excess enzyme. These ratios were determined b y measuring incorporation with various amounts of enzyme and a fixed concentration of DNA. The excess values correspond to the plateau region of this curve and the limiting to the linear portion. One unit of enzyme is defined as incorporating I nmole of labelled nucleoside triphosphate into trichloroacetic acid-precipitable material in I h (ref. I I ) . For electrophoresis of in vitro RNA an aliquot of the incubation mixture was made 0.25 % in sodium dodecyl sulphate and 0.2 M in sucrose 12. The resulting solution was then layered on top of a 2.6 % acrylamide gel and a current of 5 mA per gel applied. It was found that the protein did not cause restriction of the gel as described b y LOENING~a, either due to its low concentration or the presence of the sodium dodecyl sulphate. The gels were washed for 6 h at o ° in two changes of 5 % trichloroacetic acid to remove labelled triphosphates and then sliced into o.5-mm discs and two discs placed on a gummed label. The samples, when dry, were then stuck on aluminium planchets and counted on a Nuclear Chicago gas flow planchet counter. RNA was extracted from the gels for hybridization b y sterilely cutting out the required section from several gels and replacing them in perspex tubes. In all steps of this process great care was taken to avoid ribonuclease contamination. The RNA was then back-electrophoresed into 2 ml of electrophoresis buffer (less sodium dodecyl sulphate) contained in a dialysis bag tied over the end of the tube, the bag dipped into the lower buffer vessel thus making electrical contact. The RNA samples so obtained were bulked, made 0.5 M in NaC1 to precipitate any sodium dodecyl sulphate present and the RNA precipitated from the supernatant with 2.5 volumes of ethanol. The RNA was then dissolved in water and stored frozen. In this paper the two classes of pre-replicative early RNA will be referred to as immediate-early and delayed-early as suggested b y R. H. Epstein. The distinction between these two classes being that in vivo the synthesis of the latter class is dependent on protein synthesis. Biochim. Biophys. Acta, 2o9 (197 o) lO6-111
IO8
A . J . E . COLVILL
R E S U L T S
Fig. I shows the d i s t r i b u t i o n of label in samples t a k e n at various times from an in vitro incorporation system using limiting e n z y m e at 42° a n d then subjected to acrylamide-gel electrophoresis. After I m i n of incorporation there is a broad bim o d a l d i s t r i b u t i o n of low-molecular-weight RNA. The m a x i m u m size of the R N A increases with time a n d s i m u l t a n e o u s l y there is a shift in the t o t a l distribution. Thus those species which migrate further t h a n 4.o cm are not present in samples t a k e n after 2 min. The largest of these R N A molecules, which for convenience have been referred to as Peak I, has a molecular weight of 4.5' lO5 calculated from the d a t a of LOENING14 using R I 7 (mol. wt. I . I . I06) ts as a s t a n d a r d . At 2 m i n the m a x i m u m counts are found 2.o cm from the origin b u t some counts still r e m a i n in the P e a k - I region. As can be seen from Fig. I the position of the m a x i m a increases u n t i l at 20 m i n it is found at the origin.
(--
a(
i
'i
•
'c
(3
'
d
64 4~
)o
2o
3o
~o
so
60
70
80
io
20
30
#o
so
60
70
80
Distance moved (cm)
Fig. i. Separation of i~ vitro synthesized RNA by electrophoresis on acrylalnide gels. o.o8-ml aliquots of RNA were taken from the incubation mixture at various times: a, i min; b, 5 rain; c, IO nlin; d, 20 rain.
The disappearance of Peak I from the gel p a t t e r n with time suggested t h a t the species in this region were possibly the precursors for the larger species f o u n d between o a n d 4.o cm (referred to as P e a k II). A pulse-chase e x p e r i m e n t was therefore performed to investigate this possibility. Fig. 2 shows the p a t t e r n o b t a i n e d after a 2Biochim. Biophys. Acta, 209 (197o) lO6-111
lO9
THE FIDELITY OF TRANSCRIPTION i n vilro
Cl
o .c
E
U
10
20
30
40
50
60
70
80
10
20
30
40
5.0
60
70
8.0
Distonce moved (cm~
Fig. 2. Electrophoretic separation of pulse-chased RNA. o.o8-ml aliquots of in vitro R N A w e r e t a k e n from the incubation m i x t u r e s at 20 miD. a. Pulse-labelled w i t h 0.2/~mole/ml ~14C]ATP for 2 mid and t h e n chased w i t h 2 0 / , m o l e s / m l unlabelled A T P to m i n u t e 20 at 3 o°. b. Labelled cont i n u o u s l y for 20 mid at 3 o°.
mid pulse at 3 °0 followed by a cold chase for 18 miD. A 2D-miD incorporation at this temperature is also shown for comparison. The experiment was performed at 3 °0 in order to minimize the incorporation of label into Peak II. The RNA in the two peaks was then examined by hybridization-competition experiments to investigate their correspondence with in vivo RNA species. Labelled RNA corresponding to each of the two peaks was therefore extracted from the gels as described in MATERIALSAND METHODS, Peak I from the I-miD gel and Peak I I from the 2o-min gel. These were then competed with unlabelled in vivo RNA, isolated 2 and 12 miD after phage infection at 25 °. Fig. 3 shows that 9° % of the RNA isolated from Peak I is competed for by RNA extracted 2 min after infection. RNA extracted 12 miD after infection competes for 80 % of this RNA and for 9° % of
~o • t~ E
6o-
~0 ¸ ._E 20. U
,'0
2'0
3;°
,'0
~;0
6'0
70
Unlabelled RNA ( m g / m l )
Fig. 3. Correspondence between in vivo a n d in vitro RNA_: H y b r i d i z a t i o n competition. Competition against R N A labelled with [14C]ATP from Peaks I and I I w i t h various in vivo IZNA's. T h e hybridizations were p e r f o r m e d in the presence of 3o # g / m l T 4 DNA. H y b r i d i z a t i o n in t h e absence of unlabelled RI~A is 8o. 5 % of the i n p u t trichloroacetic acid-precipitable radioactivity for Peak I (ioo % represents 129 counts/miD) and 78.5 % for Peak I I (lOO % represents 1126 counts/miD). O , R N A from Peak I and unlabelled IRNA extracted 12 miD after infection; 0 , R N A from Peak I and unlabelled R N A extracted 2 mid after infection; 71, R N A from Peak I I and unlabelled R N A extracted 12 mid after infection; II, R N A from Peak I I and unlabelled R N A extracted 2 mid after infection.
Biochim. Biophys. Acta, 209 (197 o) i o 6 - i i i
II0
A. J . E. C O L V I L L
P e a k - I I RNA. The RNA extracted 2 min after infection only competes for approx. 15 % of the P e a k - I I RNA. The size of the larger RNA molecules synthesized in vitro in 20 rain at various temperatures with limiting enzyme increases with temperature. This can be seen from Table I which tabulates the results obtained on running these RNA samples on acrylamide gels. If the synthesis is carried out at 42o using a saturating amount of enzyme then the RNA in Peak I I migrates I.O cm which is similar to the mobility observed at 35 °. Rifampicin added 15 sec after the start of the incubation at 42°, using limiting enzyme, did not alter the size distribution of the RNA synthesized after 20 rain. TABLE
I
MIGRATION OF P E A K
I[
RNA
SYNTHESIZED
in vitro
FOR 20 MIN AT V A R I O U S T E M P E R A T U R E S
Temperature o/ synthesis (°)
Distance moved (cm)
25 3° 35 42
2.8 2.1 I.O ~ 0. I
DISCUSSION
The results of the preceding paper 1° demonstrate the synthesis of two classes of RNA during the 'early' phase of T4 infection which differ in their molecular size. T h a t equivalent to immediate-early being of molecular weight less than o.7I. lO 6 and the delayed-early being larger than this. The results presented here show that both of these classes of RNA are synthesized in vitro, however the low-molecularweight species is a precursor for the larger species. This can be seen from the disappearance of Peak I after 2 min of synthesis at 42°, as shown in Fig. I, together with the fact that these counts can be chased into the larger species (Fig. 2) and that once synthesis has commenced there are no new initiations as shown by the addition of rifampicin. The results of Table I show that as nett synthesis increases so the size of the RNA increases. This however does not hold if the synthesis is carried out in the presence of excess enzyme when the average size decreases, this is possibly due to nonspecific binding of polymerase under these conditions. Therefore although at first glance the pattern of in vitro messenger RNA synthesis appears to be similar to that observed for in vivo messenger synthesis there appear to be some basic differences. Firstly the synthesis in vivo of the larger delayedearly is dependent on protein synthesis s,l°, except perhaps for some escape synthesis which occurs after long exposures to chloramphenicol. On this basis the appearance of the larger presumably delayed-early RNA in vitro is unexpected, although this result is in agreement with previous hybridization-competition data 6,1~. Secondly the smaller RNA species synthesized in vitro appear to be precursors to the larger species. The results of Fig. 3 suggest that from the point of view of sequence specificity there is again a discrepancy between in vivo and in vitro messenger RNA. It would Biochim. Biophys. Acta,
209 (197 o) l O 6 - 1 1 1
THE FIDELITY OF TRANSCRIPTION
in vitro
III
appear that the sequences transcribed during the immediate-early phase of infection are essentially the same as those transcribed rapidly in vitro, Both delayed-early and immediate-early in vivo RNA compete almost completely for Peak-I RNA indicating that in the first minute of in vitro synthesis at 42o transcription is restricted to those sequences synthesized in vivo at the very beginning of infection. Delayed-early RNA, RNA extracted 12 rain after infection, competes for most of the RNA in Peak I I however immediate-early in vivo RNA also competes for about 15 % of this RNA. This indicates that somewhere in the order of 15 % of the in vitro P e a k - I I RNA is transcribed from sequences common to the in vivo immediate-early RNA. Approx. 75 °o is transcribed from sequences whose transcription in vivo is dependent on protein synthesis and as shown previously is not commenced until 6 rain after phage infection at 25 ° . Thus although in vitro, as in vivo, there appears to be two physically distinct classes of RNA synthesized with a temporal separation, the similarity is only superficial. I n vitro as in vivo, the immediate-early cistrons are transcribed first and the product of this synthesis is much smaller than that resulting from delayed-early transcription. The larger species synthesized in vitro however are transcribed from both immediate-early and delayed early cistrons, and all immediate-early messenger appears to end up in copolymers of immediate and delayed-early RNA, This means that the mechanism for terminating the immediate-early messengers in vivo is missing in the in vitro system. More interestingly this result also implies that the cistrons coding for immediate and delayed-early messenger must be contiguous on the T 4 genomc. ACKNOWLEDGEMENTS
I would like to thank Dr. D. G. Catcheside in whose Department this work was carried out, along with Drs. D. J. Bennett, E. H. Creaser and E. P. Geiduschek for helpful discussions and comments on this manuscript. The technical assistance ot Miss M. Colley and Mrs. J. Picker is also gratefully acknowledged. REFERENCES I ]V[. HAYASHI, M. N. }-IA.YASHI2kND S. SPIEGELMAN, Proc. Natl. Acid. Sci. U.S., 50 (1963) 664. 2 M. HAYASHI, M. N. HAYASHI AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 51 (1964) 351. 3 E. P. GEIDUSCHEK, G. P, TOCCHINI-VALENTINI AND IV[. SA.RNAT, Proc. Natl. Acad. Sci. U.S., 52 (1964) 486. 4 A. J. E. COLVILL, L. C. KANNER, G. P, TOCCHINI-VALENTINI, M. SARNAT AND E. P. GEIDUSCHEK, Proc. Natl. Acid. Sci. U.S., 53 (1965) 114o. 5 E. P. GEIDUSCHEK, Bull. Soc. Chim. Biol., 47 (I968) 1571. 6 E. P. GEIDUSCHEK, L. SNYDER, A. J. E. COLVILL AND M. SARNAT, J. Mol. Biol., 19 (1966) 541. 7 C. LEVINTHAL, J. HOSODA AND D. SHUE, in J. S. COLTER AND W. PARANCHYCH, Mo~eculat Biology o/ Viruses, Academic Press, N e w York, 1967, p. 71. 8 W. SALSER, A. BOYLE AND R. EPSTEIN, J. Mol. Biol., (197o) in the press. 9 G. MILANESI, E. N. BRODY AND E. P. GEIDUSCHEK, Nature, 221 (1967) lOI 4. io A. J. E. COLVILL, Biochim. Biophys. Acta, 209 (197o) 97. I I M. CHAMBERLIN AND P. BERG, Proc. Natl. Acid. Sci. U.S., 48 (1962) 81. 12 N. R. PACE, D. I-I. L. BISHOP AND S. SPIEGELMAN, Proc. Natl. Acid. Sci. U.S., 58 (1967) 711. 13 U. E. LOENING, Biochem. ]., lO2 (1967) 251. 14 U. E. LOENING, Biochem. J., 113 (1969) 131. 15 }~. F. GESTLAND AND H. BOEDTKER, J. Mol. Biol., 8 (1964) 496. 16 E. P. GEIDUSCHEK, E. ~ . BRODY AND D. L. WILSON, in ]3. PULMAN, Molecular Associations in Biology, Academic Press, N e w York, 1968, p. 163.
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