The largest subunit of RNA polymerase I1 in Dictyostelium: conservation of the unique tail domain and gene expression PATRICKYIP Banting and Best Department of Medical Research University of Toronto, Toronto, Ont., Canada M5G IL6
TAKYEE LAM, LAWRENCE CHAN,
AND
AND
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
CHI-HUNG SIU~ Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ont., Canada M5G IL6 Received April 15, 1992 LAM,T. Y., CHAN,L., YIP, P., and SIU,C.-H. 1992. The largest subunit of RNA polymerase I1 in Dictyostelium: conservation of the unique tail domain and gene expression. Biochem. Cell Biol. 70: 792-799. cDNAs encoding the largest subunit of RNA polymerase I1 were isolated from a Dictyostelium cDNA library. A total of 2.9 kilobases (kb) of cDNA was sequenced and the amino acid sequence of the carboxyl-terminal half of the protein was deduced. Similar to other eukaryotic RNA polymerases 11, the largest subunit of Dictyostelium RNA polymerase I1 contains a unique repetitive tail domain at its carboxyl-terminal region. It consists of 24 highly conserved heptapeptide repeats, with a consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser. In addition to the tail domain, five segments of the deduced primary structure show > 50% sequence identity with either yeast or mouse protein. RNA blots show that cDNA probes hybridized with a single mRNA species of -6 kb and immunoblots using a monoclonal antibody raised against the tail domain lighted up a single protein band of 200 kilodaltons. Interestingly, expression of the largest subunit of RNA polymerase I1 appears to be under developmental regulation. The accumulation of its mRNA showed a 60% increase during the first 3 h of development, followed by a steady decrease during the next 6 h. Cells began to accumulate a higher level of the RNA polymerase I1 mRNA after 9 h of development. When cells were treated with low concentrations of CAMPpulses to stimulate the developmental process, the pattern of mRNA accumulation moved 3 h ahead, but otherwise remained similar to that of control cells. Key words: RNA polymerase, cDNA, sequence homology, gene expression, Dictyostelium. LAM, T. Y.. CHAN,L., YIP, P., et SIU, C.-H. 1992. The largest subunit of RNA polymerase I1 in Dictyostelium: conservation of the unique tail domain and gene expression. Biochem. Cell Biol. 70 : 792-799. Les cDNA codant pour la plus grande sous-unitt de la RNA polymtrase I1 sont isoles d'une librairie de cDNA de Dictyostelium. Un total de 2,9 kilobases (kb) du cDNA sont stquenctes et la stquence des acides amints de la moitit de l'extrtmitt carboxyle de la prottine est dtduite. Semblable a la RNA polymtrase I1 d'autres eucaryotes, la plus grande sous-unitt de la RNA polymtrase I1 de Dictyostelium renferme un domaine de queue rtpttitif unique 21 sa rigion carboxyl-terminale. I1 est formt de 24 rtpetitions heptapeptidiques hautement conservtes avec une stquence consensus Tyr-Ser-Pro-Thr-Ser-Pro-Ser. En plus du domaine de queue, cinq segments de la structure primaire dtduite montrent une identitt stquentielle >50% avec la prottine de levure ou celle de souris. Les transferts RNA montrent que les sondes cDNA s'hybrident avec une seule esptce de mRNA de -6 kb et les immunotransferts utilisant un anticorps monoclonal Clevt contre le domaine de queue montrent une seule bande prottique de 200 kilodaltons. De facon inttressante, I'expression de la plus grande sous-unit6 de la RNA polymtrase I1 semble Etre sous rtgulation dheloppementale. L'accumulation de son mRNA augmente de 60% durant les trois premitres heures de dheloppement et elle diminue de facon constante durant les 6 h qui suivent. Les cellules commencent d'accumuler un taux plus tlevt du mRNA de la RNA polymtrase I1 aprts 9 h de dheloppement. Quand les cellules sont traittes avec de faibles concentrations de CAMP pour stimuler le processus dheloppemental, le profil de l'accumulation du mRNA avance de 3 h, mais pour le reste, demeure semblable a celui des cellules contrBles. Mots cl&s : RNA polymbase, cDNA, homologie stquentielle, expression gtnttique, Dictyostelium. [Traduit par la rtdaction]
Introduction Transcription in eukaryotic cells is carried out by three classes of nuclear DNA-dependent RNA polymerases (for reviews, see Sawadogo and Sentenac 1990; Woychik and Young 1990). RNA polymerase I1 is responsible for the transcription of protein-encoding genes. This pivotal role of RNA polymerase I1 in gene regulation has led to many ABBREVIATIONS: kb, kilobases(s); kDa, kilodalton(s); CTD, carboxyl-terminal domain; SDS, sodium dodecyl sulfate; SSPE, saline - sodium phosphate - EDTA buffer; SSC, 0.15 M NaCl plus 0.015 M sodium citrate; IgG, immunoglobulin G; bp, base pair@). 'present address: Department of Medicine, University of Toronto, Toronto, Ont., Canada M5S 1A8. 2 ~ u t h oto r whom all correspondence should be sent at the following address: Charles H. Best Institute, University of Toronto, 112 College Street, Toronto, Ont., Canada MSG 1L6. Printed in Canada / Imprime au Canada
recent studies on the biochemical and molecular structure of this enzyme complex. RNA polymerase I1 is composed of at least 10 subunits with a combined molecular mass of approximately 500 kDa. The two large subunits constitute almost 80% of the mass of the enzyme complex. The first RNA polymerase gene was cloned from Drosophila and corresponded to the largest subunit of RNA polymerase I1 (Searles et al. 1982). Subsequently, the gene for this subunit has been cloned from a large number of organisms (Ingles et al. 1983; Allison et al. 1985; Cho et al. 1985; Corden et al. 1985; Bartolomei et al. 1988; Smith et al. 1989; Evers et al. 1989; Li et al. 1989). Sequence data revealed an extensive homology with the /3' subunit of the Escherichia coli RNA polymerase, as well as the largest subunit of yeast RNA polymerase I (Memet et al. 1988) and RNA polymerase I11 (AUison et al. 1985). The most striking feature of the largest
LAM ET AL.
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
- .
FIG. 1. Restriction map of the partial cDNA inserts encoding the largest subunit of Dictyostelium RNA polymerase 11. The 2.9-kb cDNA segment that was sequenced is shown. Some of the unique restriction enzyme sites are indicated on top. The stippled box indicates the coding region of the cDNA and the striped box indicates the 3' untranslated sequence. The sizes and alignment of the three cDNA inserts are shown in thin black lines. RNA polymerase I1 subunit in eukaryotes is the presence of a unique tail structure or CTD, which consists of tandem repeats of a heptapeptide sequence (Allison et al. 1985,1988; Ahearn et al. 1987). The CTD structure is essential for cell viability (for a review, see Corden and Ingles 1992) and may have a crucial role in the initiation of transcription. The cellular slime mold Dictyostelium discoideurn has been used widely as a model in studies of cell differentiation and morphogenesis. In addition t o its simple life cycle, D. discoideum cells are amenable to various biochemical and genetic manipulations (for a review, see Loomis 1975). Upon the depletion of nutrients, Dictyostelium cells embark o n a developmental pathway. Cells migrate in response t o environmental CAMPto form multicellular aggregates that eventually differentiate into fruiting bodies. Extensive studies in recent years have focused o n the regulation of gene expression, since specific sets of genes are turned o n or turned off a t different stages of development. Transcription of many of the early developmental genes are known t o be either repressed or stimulated by low concentrations of cAMP administered t o cells at short intervals (Mann and Firtel 1989; Ma and Siu 1990). Therefore, studies o n the enzymatic machinery involved in transcription will be important to our understanding of the mechanisms of gene expression and cell differentiation. The RNA polymerase I1 complex has been purified from D. discoideum cells (Pong and Loomis 1973; Renart et al. 1985), but little is known about the structure of its subunits. A well-defined RNA polymerase I1 enzyme should help elucidate how this enzyme recognizes responses t o regulatory signals. In this paper, we report the cloning of cDNA for the largest subunit of RNA polymerase 11. Sequence data reveal the presence of a highly conserved CTD with 24 heptapeptide repeats. Regions of extensive sequence homology with other species are also evident. The pattern of RNA accumulation suggests that the expression of this polymerase subunit is under developmental regulation.
Materials and methods Cell strain and culture conditions The wild-type strain NC4 was cultured on agar dishes in association with Klebsiella aerogenes as the food source (Sussman 1966). Cells were grown to a density of approximately 1 x 10' cells/100-mm diameter plate and then collected for experiments. Bacteria were removed by differential centrifugation. Cells were suspended for development in 17 mM sodium phosphate potassium phosphate (pH 6.4) at 1.5 x 10' cells/mL (Cocucci and Sussman 1970). Cultures were rotated at 180 rpm on a platform shaker at room temperature. To stimulate the developmen-
tal process, cells were pulsed with cAMP at a final concentration of 2 x 10 M at 7-min intervals.
Isolation of cDNA clones A Xgtll expression library (Wong and Siu 1986), constructed from cDNA of aggregation stage cells using the method of Young and Davis (1983), was used to screen for RNA polymerase I1 cDNA. We obtained previously a small cDNA insert with sequences homologous to the CTD of the largest RNA polymerase I1 subunit of yeast when the library was screened with an antibody against ATPase. This cDNA insert was used as a probe to screen for larger inserts in the cDNA library. cDNA inserts of different sizes were isolated (see Fig. 1) and subcloned into the EcoRI site of pEMBL18 (Dente et al. 1983) for DNA sequencing. DNA sequencing DNA sequencing was performed using the dideoxy chain termination method (Sanger et al. 1977). To sequence the entire length of the cDNA clones, restriction fragments and nested deletions created by the exonuclease I11 - S1 nuclease method (Henikoff 1984) were subcloned into plasmids. Single-stranded templates were produced by infecting pEMBLl8 transformed cells with the helper phase M13K07. DNA sequencing was carried out using Sequenase (U.S. Biochemical Corporation), followed by separation on 5% polyacrylamide gels. RNA blots Total RNA was isolated from cells at different stages of development by lysing the cells with 10% SDS - 10% diethyl pyrocarbonate. This was followed by three phenol-chloroform (1:1, V/V)extractions, the last one being carried out in the presence of 0.3 M sodium acetate (pH 5). The RNA was ethanol precipitated, centrifuged, and then resuspended in water which had been previously treated with diethyl pyrocarbonate. RNA was electrophoresed on 1% agarose gels in the presence of formaldehyde. RNA was then transferred to nitrocellulose or Hybond-N membranes and hybridized to a 32~-labelled probe. Hybridization was carried out at 42°C in a solution 50% formamide, 5 x Denhardt's solution (1 070 Ficoll, 1Vo polyvinylpyrrolidone, 1% bovine serum albumin), 5 x SSPE, and 0.1% SDS, followed by washes at 6S°C in 0.1 x SSC - 0.5% SDS. For each RNA blot, the same filter was also hybridized to a D. discoideurn actin probe. Autoradiograms of different exposure times were quantified by densitometry and normalized to the amount of actin in each lane. Zrnrnunoblot analysis Total cell protein was solubilized and reduced in a mixture of 2% SDS, 8 M urea, and 2% 0-mercaptoethanol by heating at 9S°C for 5 min. Samples were electrophoresed in 7.5% polyacrylamide gel according to Laemmli (1970). Proteins were transferred electrophoretically from slab gels to nitrocellulose filters (Towbin et al. 1979). Filters were first blocked with a solution of 5% skim milk, 0.05% Tween-20, and 0.02% sodium azide in phosphate-buffered saline (pH 7.4) and then incubated with a murine anti-CTD monoclonal antibody (Moyle et al. 1989), kindly supplied by Dr. J. Ingles of the Banting and Best Department of Medical
4
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
w G C ? I A T M X : n ; G T A ~ A A ~ ~ T G C C G T ~ ~ ~ C G G T P A T A T T C A R C G T T G T C ~ T A A A A G C A A ~ ~ T C A A ~ A A A T A n 120 = A T G C C APC ~ A H G G R E G L I D T A V K T S E T G Y I Q R C L V K k M E D V S I K Y D A T V 40 A G A A A T T C ~ A n : m A ~ T T T E C C T A ~ ~ A T P X ; A ~ m ~ ~ T C M E C A T T G A T T C C c ~ A T A A T A C C G A A ~240 C G ? ] A T G R
N
S
L
G
D
V
I
Q
F
A
Y
G
E
D
G
I
P
E
C
F
V
E
N
Q
S
I
D
S
L
R
K
D
N
T
E
L
E
R
M
80
T A T F G T C A T C A A Y R H Q V D K AAATCGGAT~TTA K S D R S L L C G T A ~ G T G A ~ W R R V S D L N
G ? T G A ~ C A G A C T A T G G T G A ~ ~ ~ C A ~ A A ~ A T G ~ A G W G A I I T C A T T A P h : T C G n ; A T A360 C ~ ~ C G T A ~ P P Y G D G W M D P L V I E A V R N D S L T R D T L E K E F E R I 120 ~ ~ T T A T M C A T C T G G T C ~ A P A ~ C A ~ X C A G T G A A ~ C G T C G ~ A A T f A R T R b T G C A C W ~ A a T A T 480 'M;ATAW R N E I I P S G E A N W P L P V M L R R L I N N A Q K L T N I D I 160 C G C C A G T T G T T G T I T T A G A A A ~ ~ T ~ A C G m ~ A T T A ~ N C G C T G R T ~ C ~ T G A ~ C600 W k A W G M C T P A V V V L E I E K L V A R L K I I A T A D T T E D D E N P N R A 200 TGGGCDXJGTTT.WWTAbTGCC W ~ A ~ G A ~ A G T A C G T T C A A C A ~ A A G T A R R C G ' M ; ~ C G A A T P P A G A T T A A C ~ G C72A0 W & ~ T W A E V Y F N A T M L F S I L V R S T F A S K R V L T E F R L T E K A F L W V C 240 ~~AAA~C.prCAAGCATTCGCCCATCCAGG~TGGT~~GCACAATCAATMGTGAACCAGCAACACAAATGACACTTAATACT~ 84C0A ~ A T G E I E S K F L Q A L A H P G E M V G A L A A Q S I G E P A T Q M T L N T F R Y 280 E C ~ T A R G T A G T ~ T c ; T W A ~ ~ E C A C ~ T A k A C C ~ A T T A R C A ~ C A A A C ~ A C C A T C C C T C A C C A T C P A ~ A C A T A T 9G60G C T C G T A G V S S K N Y T L G V P R L N Q Z I N I A K Q V K T P S L T I Y L K P H H A R 320 GATATM;ATCG~rITGTAAAA~AAC~TATACTACACTTGCAAA~ARC TWXACCERRAWTkCTATWiTCC CTC 'IGAWCX AARATACCATCATCAG-T 1080 D M D R A X I V K S Q L E Y T T L A N V T S A T E I Y Y D P D P Q N T I I S E D 360 G C ~ T T T G T A A A T A G T T A T T T C G A R C T T C C A G A T G A A ~ T C G A T G T T C A T A G T A n : T C A C C ~ A C r r C G T A T C ~ ~ T A G A O G ? I A T O G T T A C C G A T ~ T T12 A0K0C A E A E F V N S Y F E L P D E E I D V H S M S P W L L R I E L D R G M V T D K K L T 400 A M G C C C A T A T C A C R l A A M T G T C G P T A ~ ' f i ~ T C ~ A A A ~ T A ~ A G ~ T G A T A A T G C C G A G A R A C ~ A T T C T T C G T A ~ G T A1320 TEGT~~CA M A D X T Q C V V R D F G L S L N C I F S D D N A E K L I L R I R M V E S Q E T 440 ~ T I C C C ~ ~ T G A T G A T G A T C A A T T C : C ~ G T C G T A W ~ T C A A A T A T G C T G A G ~ n ; G T T T T A C G T O G T A T C A A A G G T A 14 T4 ~0~ ~ C G A ~5 G 2r K G T D N D D D D Q F L R R I E S N M L S E M V L R G I K G I K K Y F M R T D E 480 P A R G A T K : C ~ C T G A A R A T X T G G T T T n ; G ~ T T C ~ T G G A T l K l T C 6 A T A C T G A ~ T T I Y : C C T C ~ A G A G G T A A T 6 A G C C A T C C ~ G T T G b T C A T ~ W G T1560 AL1C K I P K V T E N S G F G V R E E W I L D T D G V S L L E V M S R P D V D H T R T 520 $r A C C T C T A A W T A T C G ~ T C A T T C A G G T m P C X T A ~ C G T ~ W E E C E M m C W G W C G T T A T T T C C T T m W W C T A T G T M T T A W G T C 1680 AT T S N D I V E I I Q V L G I E A V R N A L L K E L R A V I S F D G S Y V N Y R H 560 ~~A~CGATG~ATGAC~hTCGTeGEATC~A~RTCACWGTCAn=GTATCARThGAG~TGGTCCACTCAn=AGATGTETTn:G 1800 AA~TGW~r
F1
2
L
A
I
L
A
D
V
M
T
Y
R
G
H
L
M
A
I
T
R
H
G
I
N
R
V
E
T
G
P
L
M
R
C
S
F
E
E
T
V
E
600
A T T C ~ h ~ C G C C A T G T n : T C ~ C T C A ~ T G ~ ~ T A P E T G A A A A T A T l l P T ~ A G G T C A A C T C C C A C C A ~ T A C n = G T A G T T T C G A A G T T T T C C E A192 A T0C A A I
L
M
D
A
A
M
F
S
E
T
D
D
V
K
G
V
T
E
N
I
I
L
G
Q
L
P
P
L
G
T
G
S
F
E
V
F
L
N
Q
640
-8
-g
10
GATATGA~~~C~GC~ACCAGRACCTTCCAA~~~ATCICAGATACTCCAGGTAGIYlWACCTTCCTATPCCTAWTGATCG2 T04 ~0 AC~WCWW D M I K N A H S I A L P E P S N V S Y P D T P G S Q T P S Y S Y G D G S T T P F 680 CATAAEC~ATGAn;CTCCATTGTCACCATWAA~CTTWCGTGGTGATTETCACCWTGCTATGARTn:ACCAGGTTA~RAATARATCCTATGGTPx=TAC.TTATCAA2160 H N P Y D A P L S P F N E T F R G D F S P S A M N S P G Y N A N K S Y G S S Y Q 720 T~TTC&CPCAATCACCAAcfiAT1Y:TCCAAC~AC:CATCCTA~TCCP9X:TPCACCATCTTA~CAAC'ITCTCCATC?PPATTCACC~CTPCAGCATCTCA~ACCAACTKIT 2280 Y F P Q S P T Y S P T S P S Y S P T S P S Y S P T S P S Y S P T S P S Y S P T S 760 C C A T C W A ~ C A A C C T C A C C A T C T T A ' S r C T C C A A C T T C A C C A T T P T A ~ W C ~ T T C ~ C T T C C T ~ T C A C C X T T C A C C A T C A T A C T C C C C ~ T C A C C A T C A T 24 A ~0C0A P S Y S P T S P S Y S P T S P P Y S P T S P S Y S P T S P S Y S P T S P S Y S P 800 A C C T C P v C C A T C T l l A C T C A C C A A C C ~ F X ! C C T C C T A T P C A C C A A C ~ C A T C R T A ~ A C C A A C ~ C A T C A T A ~ A C C A A C T T C ~ C A T C A T A C T C T C C ~ T P C A C C A2520 TC~AT T S F S Y S P T S P S Y S P T S F S Y S P T S P S Y S P T S P S Y S P T S P S Y 840 ~ ~ C A A C C T C T C C ~ A T ~ T C A C C A A C C T C A C C A T C T T A ~ C A R C C T C A C C A T C ~ A ~ A C C A T C R . ~ C C C A ~ ~ A ' f i c A C C A A G ~ G C C A T C A T A ~ A l : C A2640 AGTTCPI:CA S P T S P S Y S P T S P S Y S P T S P S Y S P S S P S Y S P S S P S Y S P S S P 880 TCATACTCACCATCATWCAACCTTP~AAACARATAT~TA~AACCARATAAT~AREAACATTAAGATACCATTTCCTATTIETRARTATTTAA~TATATAR 2760 AT S Y S P S S P T F T N K Y N Y Q P N N K K K 902 ~ ~ W n a A C A R A T T A A G W T A T ~ ~ P T A R T A A A ~ ~ A 2874 T A C X
FIG. 2. Nucleotide sequence of the cDNA coding for the Dictyostelium RNA polymerase I1 subunit. Nucleotides are numbered at the right in 5' to 3' direction. The deduced amino acid sequence is shown in single letter code below the nucleotide sequence and is numbered also at the right. The termination codon is in bold type and the three putative polyaclenylntion signals are underlined.
LAM ET AL.
Region I
D.d.
Mouse Yeast
?!BY#WS=%C
D@Y@A~~@EIBGc
60 909 ~ G G R E G L ~ Z T r A V I Z T ~ T G Y I Q ~ V ~ E D I M V H Y D N T T R W S L G ~ X Q P I Y G E D G M876D A A
ST
ANGGREGLIDTAVKTAETGYIQR~SLIXSM-ES~YDATVRNSINQWQLRYGEDGLAGE
*NegrBs~KDfiTELrny#l
82
~ ~ F Q M C A P L K P ~ ~ E K I 921 FFR HIEKQSLD~I G G ~ D W E 898 ~ Region X I
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
D.d. Mouse Yeast
~
S
~+T~@E%~H~&V
%
E
IE@
176
b ~ m ~ - V I ~ G D S K W L P C N L L R M I W A Q K I F & N P R ~ ; ~ S D L H P I ~ E1016 ~EL 1
D R K F L R - E V P V D G E ~ P L P W T R I U : XQNAQQTPHIDHTKPSDLTIKDIVLGVKDL 993
Region I11
B K ~ T ~ ~ @ E & ~ c
gT
D.d.
&!&I@S
Mouse Yeast
M A ' P L L L F N I H ~ S ~ C S ~ M A E E P ~ S G E A F D ~ ~ ; G E X E S K P N W A H P G E 1094 NVG~QS DAVTLPCCL;L;RSRE~TXIRVLQEY RLrTRQAFDWVfrSHTEAQPLRS SWHPGEMVGVWIQS 1071
265
'RIVK&&EY k%%iWMsAkkfWWd@dNCfl~ f SWAEFWS W&~,&E~IDVHSM--~PR~ 383 K D ? L C R L E ~ ' ~ K ~ P T A N T A I Y Y D P N P Q S T ~ A E : D Q ~ Y Y E M P D F D V A R 1210 ----SPW KLTRSAXEBTTLKSVTIASE XYYDPDPRSTVI PEDEEI IQLHFSLLDsEAEQSFDQB6'Pw
1191
Region IV
D. d. Mouse Yeast
2
~
~
@
~
@
~
476
1 , 1284
#
#
~
~
~
~
~
~
~
f
k
~ , Q ~ ~ v ; F ~ ~ c ~ ~ ~ ~ ~ SDWMY $ L 1309Q G I ~ Q I
EE$HMBKKFY$NT#~NI ,
,
50% sequence identity. The degree of homology is considerably higher if conserved amino acid changes are included in the calculation. These five conserved regions (I to V) consist of 82, 56, 228, 30, and 141 amino acids, respectively. In region I, the percentage of sequence identity is 63% with both mouse and yeast. Amino acids in region I1 show 50% identity with mouse and 55% identity with yeast. Interestingly, in regions 111-V, the percentages
of sequence identity with mouse (66, 67, and 73'70, respectively) are considerably higher than those with yeast (60,40, and 62070, respectively). Within regions I, 111, and V, there are short segments that show >80% sequence identity with either mouse or yeast. The five conserved regions are separated by segments of highly variable amino acid sequences. Computer search also revealed that the Dictyostelium sequence is homologous to the large subunit of other RNA polymerases, but to a lesser extent than the RNA polymerase I1 subunit. The most notable homologous sequence is the tandemly repeated heptapeptide at the carboxyl terminus. This tail structure is unique to the largest subunit of RNA polymerase 11, thus further confirming the identity of our cDNA clones. The CTD of the Dictyostelium polymerase subunit is composed of 24 heptapeptide repeats, with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser(Fig. 4). The same heptapeptide sequence is repeated 26 times in yeast (Allison et al. 1985) and 52 times in mouse (Ahearn et al. 1987). Variations from the consensus sequence occur to different degrees in the CTD of a number of species. In the case of Dictyostelium, there are only nine substitutions in the entire CTD domain, with one in position two, five in position four, and three in position seven, and with six of these changes being conservative. The last heptapeptide repeat is followed by a variable sequence of 14 amino acids, with three Lys residues ending at the carboxyl terminus. Gene expression during development In RNA blots, a labelled cDNA probe coding for the CTD hybridized with a single mRNA species of -6 kb (Fig. 5), which is sufficient to code for a protein of 200 kDa. The presence of an RNA polymerase I1 subunit with the unique
797
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
LAM ET AL.
FIG. 5. RNA blot and immunoblot of the largest RNA polymerase I1 subunit. Vegetative Dictyostelium cells were collected and total RNA was isolated and separated on agarose gel. In A, the RNA blot was hybridized with a 32~-labelledcDNA probe (the EcoRI fragment at the 3 ' end of the cDNA). The probe hybridized with a single RNA species of -6 kb (arrow). The positions of the 26s and 17s rRNAs are indicated on the left. Cells were also solubilized in SDS for gel electrophoresis. In B, proteins were indirectly stained with a monoclonal antibody directed against the CTD of RNA polymerase I1 largest subunit (lane a). The antibody reacted with a single protein band at 200 kDa (arrow). A similar sample reacted with preimmune serum is shown in lane b.
tail structure in Dictyostelium cells was ascertained by immunoblots. Total cellular protein derived was stained indirectly with a monoclonal antibody raised against the CTD heptapeptide sequence (Moyle et al. 1989). It reacted specifically with a protein band of -200 kDa (Fig. 5), a molecular size consistent with that of the largest subunit of RNA polymerase I1 subunit in Dictyostelium (Renart et al. 1985). To examine the expression of the largest RNA polymerase I1 subunit during development, RNA samples were isolated from cells at different developmental stages and probed with the same cDNA fragment (Fig. 6). The mRNA level increased by -60% at 3 h of development and then decreased steadily to about half the amount in vegetative cells in the next 6 h. This was followed by an accumulation of RNA transcripts between 9 and 15 h, resulting in a 3.5-fold increase in mRNA level. When cells were treated with low doses of CAMP, the general pattern of mRNA accumulation was moved forward by about 3 h. Interestingly, treating of cells with cAMP did not stimulate an increase in mRNA accumulation, but rather led to an overall reduction in mRNA level. Discussion We have cloned and sequenced cDNA coding for the carboxyl-terminal half of the largest subunit of RNA polymerase I1 from D. discoideum. The cDNA hybridized with a single mRNA species of -6 kb, sufficient to code for a protein of 200 kDa, and the amino acid sequence deduced from the cDNA sequence showed extensive sequence identity with the same RNA polymerase I1 subunit
Time of development 6. Accumulation of RNA polymerase I1 subunit mRNA during development. Vegetative cells were collected for development either in the absence of cAMP or in the presence of cAMP pulses at 7-min intervals, giving a final cAMP concentration of M. Cells were collected every 3 h during development 2 x and total RNA was isolated and separated on agarose gels. Blots were probed with a 32~-labelledcDNA probe. The same blots were also probed with actin cDNA. Autoradiograms were quantitated by densitometry and the values were normalized against the actin content. The relative levels of mRNA were estimated for samples collected at different time points, with stippled bars for the nonpulsed control and the striped bars for the CAMP-pulsed sample. Values represent the average of two experiments. FIG.
in many different species. Sequence analysis reveals that the RNA polymerase subunits are a group of highly conserved proteins. Sequences of the largest RNA polymerase I1 subunit of Saccharomyces cerevisiae (Allison et al. 1985), Caenorhabditis elegans (Bird and Riddle 1989), Drosophila melanogaster (Jokerst et al. 1989), and the mouse (Ahearn et al. 1987) show that almost 40% of the amino acid residues are invariant. Interestingly, many of the conditional mutations of RNA polymerase I1 affect these invariant amino acid residues (Scafe et al. 1990), suggesting that the conserved segments of the protein may have important structural or functional roles. A remarkable feature of the eukaryotic RNA polymerase I1 is the presence of a heptapeptide repeat in the CTD of its largest subunit. The Dictyostelium subunit has a CTD consisting of 24 repeats of the consensus sequence: Tyr-SerPro-Thr-Ser-Pro-Ser. This consensus sequence is repeated 52 times in mouse (Corden et al. 1985) and hamster (Allison et a1 1988), 42-44 times in Drosophila (Allison et al. 1988; Zehring et al. 1988), 32 times in C. elegans (Bird and Riddle 1989), 26-27 times in yeast (Allison et al. 1985), and 17 times in Plasmodium falciparum (Li et al. 1989). The length of the CTD seems to be related to the complexity of the organism. The amino acids in positions one (Tyr), three (Pro), and six (Pro) are nearly invariant, while the amino acid at position seven shows a high degree of divergence. The Dictyostelium CTD is highly conserved, with 18 exact matches out of 24 repeats. In comparison, yeast has 17 exact matches out of 26, while Drosophila has the most divergent
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
798
BIOCHEM. CELL BIOL. VOL. 70,
CTD, with only 2 of the 42-44 repeats matching the consensus sequence. The CTD of RNA polymerase I1 is likely to be fully exposed to the solvent and project out from the globular fold of the rest of the polypeptide, since all the amino acid side chains, excepting that of proline, are hydrophilic. The repeated pattern of this domain also suggests a regular, flexible structure. Genetic studies show that deletions of all or most of the repeats in the yeast or mouse CTD are lethal (Allison et al. 1988; Zehring et al. 1988). These results imply that the CTD has an essential role in transcription. However, the precise function of the CTD is still unknown. It is possible that the extended CTD may be involved in interaction with certain transcriptional factors at a distance (Corden and Ingles 1992), thus facilitating the recruitment of trans-acting factors to the transcription initiation complex. However, the nature of such target proteins remains to be identified. Another possibility is that the CTD may participate in transcription initiation through direct interaction with DNA (Suzuki 1990). In both models, the role of the CTD in transcriptional regulation may be mediated by phosphorylation and dephosphorylation reactions, since the CTD is known to be heavily phosphorylated in vivo (Cadena and Dahmus 1987). The expression of RNA polymerase I1 is essential for growth and development and is expected to behave like a housekeeping gene. However, our results show that the expression of the largest subunit of RNA polymerase I1 is under developmental regulation. During the first 3 h of Dictyostelium development, its mRNA level rose slightly and then decreased steadily for the next 6 h. This corresponded to a period when the number of mRNA species does not change significantly (Blumberg and Lodish 1980). Although the expression of some genes are induced, the number of such genes is relatively low (Alton and Lodish 1977; Mangiarotti et al. 1983). The metabolic activity of cells during this initial period of starvation is apparently lower and a decrease in the overall synthesis of glycoprotein has been observed (Lam and Siu 1981). By contrast, about 2500 new species of mRNA are synthesized in postaggregation cells (Blumberg and Lodish 1980). The transcription of this large number of developmentally regulated genes is preceded by an increase in the mRNA level of the largest subunit of RNA polymerase 11, which begins at the late-aggregation stage. A close temporal correlation therefore exists between the increase in RNA polymerase I1 expression and the elevated transcriptional activities in the postaggregation stages of development. Many genes expressed during the aggregation and postaggregation stages are under the control of CAMP.A number of early genes are sensitive to pulses of low concentrations of CAMP,while the postaggregation stage genes require a constant level of much higher concentration of cAMP for stimulation (for a review, see Firtel 1991). However, cAMP does not appear to have a major effect on the mRNA level of the largest subunit of RNA polymerase 11, except that the pattern of mRNA levels was shifted 3 h forward. This is consistent with the fact that exogenously added cAMP can accelerate the developmental program by 3-4 h. cAMP also caused an overall reduction in the mRNA level of the RNA polymerase I1 subunit. Whether this is due to a decrease in mRNA stability or a general reduction in the transcription of housekeeping genes is not clear. This unique
1992
pattern of expression of the largest subunit of RNA polymerase I1 suggests a regulatory mechanism differing from those involved in the expression of stage-specific genes. Further analysis of its gene and promoter region will be required to understand the regulation of its expression. Acknowledgements We thank Dr. Jim Ingles for his advice and his generous gift of monoclonal antibody directed against the CTD of the largest subunit of RNA polymerase 11. This work was supported by an operating grant from the Medical Research Council of Canada and L. Chan was supported by a summer studentship from the Life Sciences Committee, University of Toronto. Ahearn, J.M., Bartolomei, MS., West, M.L., Cisek, L.J., and Corden, J.L. 1987. Cloning and sequence analysis of the mouse genomic locus encoding the largest subunit of RNA polymerase 11. J. Biol. Chem. 262: 10 695 - 10 705. Allison, L.A., Moyle, M., Shales, M., and Ingles, C.J. 1985. Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases. Cell, 42: 599-610. Allison, L.A., Wong, J.K., Fitzpatrick, D., Moyle, M., and Ingles, C.J. 1988. The C-terminal domain of the largest subunit of RNA polymerase I1 of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: a conserved structure with an essential function. Mol. Cell. Biol. 8: 321-329. Alton, T.H., and Lodish, H.F. 1977. Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum. Dev. Biol. 60: 180-206. Bartolomei, M.S., Halden, N.F., Cullen, C.R., and Corden, J .L. 1988. Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase 11. Mol. Cell. Biol. 8: 330-339. Bird, D.M., and Riddle, D.L. 1989. Molecular cloning and sequencing of ama-1, the gene encoding the largest subunit of Caenorhabditis elegans RNA polymerase 11. Mol. Cell. Biol. 9: 4119-4130.
Blumberg, D.D., and Lodish, H.F. 1980. Changes in the messenger RNA population during differentiation of Dictyostelium discoideum. Dev. Biol. 78: 285-300. Cadena, D.L., and Dahmus, M.E. 1987. Messenger RNA synthesis in mammalian cells is catalyzed by the phosphorylation form of RNA polymerase 11. J. Biol. Chem. 262: 12 468 - 12 474. Cho, K.W.Y.. Khalili, K., Zandomeni, R., and Weinmann, R. 1985. The gene encoding the largest subunit of human RNA polymerase 11. J. Biol. Chem. 260: 15 204 - 15 210. Cocucci, S., and Sussman, M. 1970. RNA in cytoplasmic and nuclear fractions of cellular slime mold amebas. J. Cell Biol. 45: 399-407. Corden, J.L., and Ingles, C.J. 1992. The carboxyl-terminaldomain of the largest subunit of eucaryotic RNA polymerase 11. In Transcriptional regulation. Edited by K.R. Yamamoto and S. McKnight. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. In press. Corden, J.L., Cadena, D.L., Aheam, J.M., and Dahmus, M.E. 1985. A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase 11. Proc. Natl. Acad. Sci. U.S.A. 82: 7934-7938. Dente, L., Cesareni, G., and Cortese, R. 1983. pEMBL: a new family of single stranded plasmids Nucleic Acids Res. 11: 1645-1655.
Evers, R., Hammer, A., Kock, J., Jess, W., Borst, P., Memet, S., and Cornelissen, A. W. 1989. Trypanosoma brucei contains two RNA polymerase I1 largest subunit genes with an altered C-terminal domain. Cell, 56: 585-597.
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
LAM ET AL.
Firtel, R.A. 1991. Signal transduction pathways controlling multicellular development in Dictyostelium. Trends Genet. 7 : 381-388. Henikoff, S. 1984. Unidirectional digestion with exonuclease I11 creates targeted breakpoints for DNA sequencing. Gene, 28: 351-359. Ingles, C. J., Biggs, J., Wong, J.K.-C., Weeks, J.R., and Greenleaf, A.L. 1983. Identification of a structural gene for an RNA polymerase I1 polypeptide in Drosophila melanogaster and marnmalian species. Proc. Natl. Acad. Sci. U.S.A. 80: 8896-3400. Jokerst, R.S., Weeks, J.R., Zehring, W.A., and Greenleaf, A.L. 1989. Analysis of the gene encoding the largest subunit of RNA polymerase I1 in Drosophila. Mol. Gen. Genet. 215: 266-275. Kimmel, A., and Firtel, R.A. 1983. Sequence organization in Dictyostelium: unique structure at the 5'-ends of protein coding genes. Nucleic Acids Res. 11: 541-552. Laemmli, V.K. 1970. Cleavage of structural proteins during assembly of the bacteriophage T4. Nature (London), 227: 680-685. Lam, T.Y., and Siu, C.-H. 1981. Synthesis of stage-specific glycoproteins in Dictyostelium discoideum. Dev. Biol. 83: 127-137. Li, W.B., Bzik, D.J., Gu, H., Tanaka, M., Fox, B.A., and Inselburg, J. 1989. An enlarged largest subunit of Plasmodium falc@arum RNA polymerase I1 defines conserved and variable RNA polymerase domains. Nucleic Acids Res. 17: 9621-9636. Loomis, W.F. 1975. Dictyostelium discoideum: a developmental system. Academic Press, Inc., New York. pp. 1-85. Ma, P.C.-C., and Siu, C.-H. 1990. A pharmacologically distinct cyclic AMP receptor is responsible for the regulation of gp80 expression in Dictyostelium discoideum. Mol. Cell. Biol. 7 : 3297-3306. Mangiarotti, G., Bozzaro, S., Landfear, S., and Lodish, H.F. 1983. Cell-cell contact, CAMP, and gene expression during development of Dictyostelium discoideum. Curr. Top. Dev. Biol. 18: 117-154. Mann, S.K.O., and Firtel, R.A. 1989. Two-phase regulatory pathway controls CAMPreceptor-mediated expression of early genes in Dictyostelium. Proc. Natl. Acad. Sci. U.S.A. 86: 1924- 1928. Memet, S., Gouy, M., Marck, C., Sentenac, A., and Buhler, J.-M. 1988. The gene coding for the largest subunit of yeast RNA polymerase A. J. Biol. Chem. 263: 2830-2839. Moyle, M., Lee, J.S., Anderson, W.F., and Ingles, C.J. 1989. The C-terminal domain of the largest subunit of RNA polymerase I1 and transcription initiation. Mol. Cell. Biol. 9: 5750-5753. Pong, S., and Loomis, W.F. 1973. Multiple nuclear RNA polyrnerases during development of Dictyostelium discoideum. J. Biol. Chem. 248: 3933-3939. Proudfoot, N.J., and Brownlee, G.G. 1976. 3' non-coding region
799
sequences in eukaryotic messenger RNA. Nature (London), 263: 21 1-214. Renart, M.F., Sastre, L., Diaz, V., and Sebastian, J. 1985. Purification and subunit structure of RNA polymerase I and I1 from Dictyostelium discoideum vegetative cells. Mol. Cell. Biochem. 66: 21-29. Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467. Sawadogo, M., and Sentenac, A. 1990. RNA polymerase B (11) and general transcription factors. Annu. Rev. Biochem. 59: 71 1-754. Scafe, C., Nonet, M., and Young, R.A. 1990. RNA polymerase I1 mutants defective in transcription of a subset of genes. Mol. Cell. Biol. 10: 1010-1016. Searles, L.L., Jokerst, R.S., Bingham, P.M., Voekler, R.A., and Greenleaf, A.L. 1982. Molecular cloning of sequences from a Drosophila RNA polymerase I1 locus by P element transponson tagging. Cell, 31: 585-592. Smith, J.L., Levin, J.R., Ingles, C.J., and Agabian, N. 1989. In trypanosomes the homolog of the largest subunit of RNA polymerase I1 is encoded by two genes and has a highly unusual C-terminal domain structure. Cell, 56: 815-827. Sussman, M. 1966. Biochemical and genetic methods in the study of cellular slime mold development. Methods Cell Physiol. 2: 397-409. Suzuki, M. 1990. The heptad repeat in the largest subunit of RNA polymerase I1 binds by intercalating into DNA. Nature (London), 344: 562-565. Towbin, H., Staehelin, T., and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354. Warrick, H.M., and Spudich, J.A. 1988. Codon preference in Dictyostelium discoideum. Nucleic Acids Res. 16: 6617-6635. Wong, L.M., and Siu, C.-H. 1986. Cloning of cDNA for the contact site A glycoprotein of Dictyostelium discoideum. Proc. Natl. Acad. Sci. U.S.A. 83: 4248-4252. Woychik, N.A., and Young, R.A. 1990. RNA polymerase 11: subunit structure and function. Trends Biochem. Sci. 15: 347-350. Young, R.A., and Davis, R.W. 1983. Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. U.S.A. 80: 1194-1 198. Zehring, W.A., Lee, J.M., Weeks, J.R., Jokerst, R.S., and Greenleaf, A.L. 1988. the C-terminal repeat domain of RNA polymerase I1 largest subunit is essential in vivo but is not required for accurate transcription in vitro. Proc. Natl. Acad. Sci. U.S.A. 85: 3698-3702.
TAKYEE LAM, LAWRENCE CHAN,
AND
AND
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
CHI-HUNG SIU~ Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ont., Canada M5G IL6 Received April 15, 1992 LAM,T. Y., CHAN,L., YIP, P., and SIU,C.-H. 1992. The largest subunit of RNA polymerase I1 in Dictyostelium: conservation of the unique tail domain and gene expression. Biochem. Cell Biol. 70: 792-799. cDNAs encoding the largest subunit of RNA polymerase I1 were isolated from a Dictyostelium cDNA library. A total of 2.9 kilobases (kb) of cDNA was sequenced and the amino acid sequence of the carboxyl-terminal half of the protein was deduced. Similar to other eukaryotic RNA polymerases 11, the largest subunit of Dictyostelium RNA polymerase I1 contains a unique repetitive tail domain at its carboxyl-terminal region. It consists of 24 highly conserved heptapeptide repeats, with a consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser. In addition to the tail domain, five segments of the deduced primary structure show > 50% sequence identity with either yeast or mouse protein. RNA blots show that cDNA probes hybridized with a single mRNA species of -6 kb and immunoblots using a monoclonal antibody raised against the tail domain lighted up a single protein band of 200 kilodaltons. Interestingly, expression of the largest subunit of RNA polymerase I1 appears to be under developmental regulation. The accumulation of its mRNA showed a 60% increase during the first 3 h of development, followed by a steady decrease during the next 6 h. Cells began to accumulate a higher level of the RNA polymerase I1 mRNA after 9 h of development. When cells were treated with low concentrations of CAMPpulses to stimulate the developmental process, the pattern of mRNA accumulation moved 3 h ahead, but otherwise remained similar to that of control cells. Key words: RNA polymerase, cDNA, sequence homology, gene expression, Dictyostelium. LAM, T. Y.. CHAN,L., YIP, P., et SIU, C.-H. 1992. The largest subunit of RNA polymerase I1 in Dictyostelium: conservation of the unique tail domain and gene expression. Biochem. Cell Biol. 70 : 792-799. Les cDNA codant pour la plus grande sous-unitt de la RNA polymtrase I1 sont isoles d'une librairie de cDNA de Dictyostelium. Un total de 2,9 kilobases (kb) du cDNA sont stquenctes et la stquence des acides amints de la moitit de l'extrtmitt carboxyle de la prottine est dtduite. Semblable a la RNA polymtrase I1 d'autres eucaryotes, la plus grande sous-unitt de la RNA polymtrase I1 de Dictyostelium renferme un domaine de queue rtpttitif unique 21 sa rigion carboxyl-terminale. I1 est formt de 24 rtpetitions heptapeptidiques hautement conservtes avec une stquence consensus Tyr-Ser-Pro-Thr-Ser-Pro-Ser. En plus du domaine de queue, cinq segments de la structure primaire dtduite montrent une identitt stquentielle >50% avec la prottine de levure ou celle de souris. Les transferts RNA montrent que les sondes cDNA s'hybrident avec une seule esptce de mRNA de -6 kb et les immunotransferts utilisant un anticorps monoclonal Clevt contre le domaine de queue montrent une seule bande prottique de 200 kilodaltons. De facon inttressante, I'expression de la plus grande sous-unit6 de la RNA polymtrase I1 semble Etre sous rtgulation dheloppementale. L'accumulation de son mRNA augmente de 60% durant les trois premitres heures de dheloppement et elle diminue de facon constante durant les 6 h qui suivent. Les cellules commencent d'accumuler un taux plus tlevt du mRNA de la RNA polymtrase I1 aprts 9 h de dheloppement. Quand les cellules sont traittes avec de faibles concentrations de CAMP pour stimuler le processus dheloppemental, le profil de l'accumulation du mRNA avance de 3 h, mais pour le reste, demeure semblable a celui des cellules contrBles. Mots cl&s : RNA polymbase, cDNA, homologie stquentielle, expression gtnttique, Dictyostelium. [Traduit par la rtdaction]
Introduction Transcription in eukaryotic cells is carried out by three classes of nuclear DNA-dependent RNA polymerases (for reviews, see Sawadogo and Sentenac 1990; Woychik and Young 1990). RNA polymerase I1 is responsible for the transcription of protein-encoding genes. This pivotal role of RNA polymerase I1 in gene regulation has led to many ABBREVIATIONS: kb, kilobases(s); kDa, kilodalton(s); CTD, carboxyl-terminal domain; SDS, sodium dodecyl sulfate; SSPE, saline - sodium phosphate - EDTA buffer; SSC, 0.15 M NaCl plus 0.015 M sodium citrate; IgG, immunoglobulin G; bp, base pair@). 'present address: Department of Medicine, University of Toronto, Toronto, Ont., Canada M5S 1A8. 2 ~ u t h oto r whom all correspondence should be sent at the following address: Charles H. Best Institute, University of Toronto, 112 College Street, Toronto, Ont., Canada MSG 1L6. Printed in Canada / Imprime au Canada
recent studies on the biochemical and molecular structure of this enzyme complex. RNA polymerase I1 is composed of at least 10 subunits with a combined molecular mass of approximately 500 kDa. The two large subunits constitute almost 80% of the mass of the enzyme complex. The first RNA polymerase gene was cloned from Drosophila and corresponded to the largest subunit of RNA polymerase I1 (Searles et al. 1982). Subsequently, the gene for this subunit has been cloned from a large number of organisms (Ingles et al. 1983; Allison et al. 1985; Cho et al. 1985; Corden et al. 1985; Bartolomei et al. 1988; Smith et al. 1989; Evers et al. 1989; Li et al. 1989). Sequence data revealed an extensive homology with the /3' subunit of the Escherichia coli RNA polymerase, as well as the largest subunit of yeast RNA polymerase I (Memet et al. 1988) and RNA polymerase I11 (AUison et al. 1985). The most striking feature of the largest
LAM ET AL.
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
- .
FIG. 1. Restriction map of the partial cDNA inserts encoding the largest subunit of Dictyostelium RNA polymerase 11. The 2.9-kb cDNA segment that was sequenced is shown. Some of the unique restriction enzyme sites are indicated on top. The stippled box indicates the coding region of the cDNA and the striped box indicates the 3' untranslated sequence. The sizes and alignment of the three cDNA inserts are shown in thin black lines. RNA polymerase I1 subunit in eukaryotes is the presence of a unique tail structure or CTD, which consists of tandem repeats of a heptapeptide sequence (Allison et al. 1985,1988; Ahearn et al. 1987). The CTD structure is essential for cell viability (for a review, see Corden and Ingles 1992) and may have a crucial role in the initiation of transcription. The cellular slime mold Dictyostelium discoideurn has been used widely as a model in studies of cell differentiation and morphogenesis. In addition t o its simple life cycle, D. discoideum cells are amenable to various biochemical and genetic manipulations (for a review, see Loomis 1975). Upon the depletion of nutrients, Dictyostelium cells embark o n a developmental pathway. Cells migrate in response t o environmental CAMPto form multicellular aggregates that eventually differentiate into fruiting bodies. Extensive studies in recent years have focused o n the regulation of gene expression, since specific sets of genes are turned o n or turned off a t different stages of development. Transcription of many of the early developmental genes are known t o be either repressed or stimulated by low concentrations of cAMP administered t o cells at short intervals (Mann and Firtel 1989; Ma and Siu 1990). Therefore, studies o n the enzymatic machinery involved in transcription will be important to our understanding of the mechanisms of gene expression and cell differentiation. The RNA polymerase I1 complex has been purified from D. discoideum cells (Pong and Loomis 1973; Renart et al. 1985), but little is known about the structure of its subunits. A well-defined RNA polymerase I1 enzyme should help elucidate how this enzyme recognizes responses t o regulatory signals. In this paper, we report the cloning of cDNA for the largest subunit of RNA polymerase 11. Sequence data reveal the presence of a highly conserved CTD with 24 heptapeptide repeats. Regions of extensive sequence homology with other species are also evident. The pattern of RNA accumulation suggests that the expression of this polymerase subunit is under developmental regulation.
Materials and methods Cell strain and culture conditions The wild-type strain NC4 was cultured on agar dishes in association with Klebsiella aerogenes as the food source (Sussman 1966). Cells were grown to a density of approximately 1 x 10' cells/100-mm diameter plate and then collected for experiments. Bacteria were removed by differential centrifugation. Cells were suspended for development in 17 mM sodium phosphate potassium phosphate (pH 6.4) at 1.5 x 10' cells/mL (Cocucci and Sussman 1970). Cultures were rotated at 180 rpm on a platform shaker at room temperature. To stimulate the developmen-
tal process, cells were pulsed with cAMP at a final concentration of 2 x 10 M at 7-min intervals.
Isolation of cDNA clones A Xgtll expression library (Wong and Siu 1986), constructed from cDNA of aggregation stage cells using the method of Young and Davis (1983), was used to screen for RNA polymerase I1 cDNA. We obtained previously a small cDNA insert with sequences homologous to the CTD of the largest RNA polymerase I1 subunit of yeast when the library was screened with an antibody against ATPase. This cDNA insert was used as a probe to screen for larger inserts in the cDNA library. cDNA inserts of different sizes were isolated (see Fig. 1) and subcloned into the EcoRI site of pEMBL18 (Dente et al. 1983) for DNA sequencing. DNA sequencing DNA sequencing was performed using the dideoxy chain termination method (Sanger et al. 1977). To sequence the entire length of the cDNA clones, restriction fragments and nested deletions created by the exonuclease I11 - S1 nuclease method (Henikoff 1984) were subcloned into plasmids. Single-stranded templates were produced by infecting pEMBLl8 transformed cells with the helper phase M13K07. DNA sequencing was carried out using Sequenase (U.S. Biochemical Corporation), followed by separation on 5% polyacrylamide gels. RNA blots Total RNA was isolated from cells at different stages of development by lysing the cells with 10% SDS - 10% diethyl pyrocarbonate. This was followed by three phenol-chloroform (1:1, V/V)extractions, the last one being carried out in the presence of 0.3 M sodium acetate (pH 5). The RNA was ethanol precipitated, centrifuged, and then resuspended in water which had been previously treated with diethyl pyrocarbonate. RNA was electrophoresed on 1% agarose gels in the presence of formaldehyde. RNA was then transferred to nitrocellulose or Hybond-N membranes and hybridized to a 32~-labelled probe. Hybridization was carried out at 42°C in a solution 50% formamide, 5 x Denhardt's solution (1 070 Ficoll, 1Vo polyvinylpyrrolidone, 1% bovine serum albumin), 5 x SSPE, and 0.1% SDS, followed by washes at 6S°C in 0.1 x SSC - 0.5% SDS. For each RNA blot, the same filter was also hybridized to a D. discoideurn actin probe. Autoradiograms of different exposure times were quantified by densitometry and normalized to the amount of actin in each lane. Zrnrnunoblot analysis Total cell protein was solubilized and reduced in a mixture of 2% SDS, 8 M urea, and 2% 0-mercaptoethanol by heating at 9S°C for 5 min. Samples were electrophoresed in 7.5% polyacrylamide gel according to Laemmli (1970). Proteins were transferred electrophoretically from slab gels to nitrocellulose filters (Towbin et al. 1979). Filters were first blocked with a solution of 5% skim milk, 0.05% Tween-20, and 0.02% sodium azide in phosphate-buffered saline (pH 7.4) and then incubated with a murine anti-CTD monoclonal antibody (Moyle et al. 1989), kindly supplied by Dr. J. Ingles of the Banting and Best Department of Medical
4
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
w G C ? I A T M X : n ; G T A ~ A A ~ ~ T G C C G T ~ ~ ~ C G G T P A T A T T C A R C G T T G T C ~ T A A A A G C A A ~ ~ T C A A ~ A A A T A n 120 = A T G C C APC ~ A H G G R E G L I D T A V K T S E T G Y I Q R C L V K k M E D V S I K Y D A T V 40 A G A A A T T C ~ A n : m A ~ T T T E C C T A ~ ~ A T P X ; A ~ m ~ ~ T C M E C A T T G A T T C C c ~ A T A A T A C C G A A ~240 C G ? ] A T G R
N
S
L
G
D
V
I
Q
F
A
Y
G
E
D
G
I
P
E
C
F
V
E
N
Q
S
I
D
S
L
R
K
D
N
T
E
L
E
R
M
80
T A T F G T C A T C A A Y R H Q V D K AAATCGGAT~TTA K S D R S L L C G T A ~ G T G A ~ W R R V S D L N
G ? T G A ~ C A G A C T A T G G T G A ~ ~ ~ C A ~ A A ~ A T G ~ A G W G A I I T C A T T A P h : T C G n ; A T A360 C ~ ~ C G T A ~ P P Y G D G W M D P L V I E A V R N D S L T R D T L E K E F E R I 120 ~ ~ T T A T M C A T C T G G T C ~ A P A ~ C A ~ X C A G T G A A ~ C G T C G ~ A A T f A R T R b T G C A C W ~ A a T A T 480 'M;ATAW R N E I I P S G E A N W P L P V M L R R L I N N A Q K L T N I D I 160 C G C C A G T T G T T G T I T T A G A A A ~ ~ T ~ A C G m ~ A T T A ~ N C G C T G R T ~ C ~ T G A ~ C600 W k A W G M C T P A V V V L E I E K L V A R L K I I A T A D T T E D D E N P N R A 200 TGGGCDXJGTTT.WWTAbTGCC W ~ A ~ G A ~ A G T A C G T T C A A C A ~ A A G T A R R C G ' M ; ~ C G A A T P P A G A T T A A C ~ G C72A0 W & ~ T W A E V Y F N A T M L F S I L V R S T F A S K R V L T E F R L T E K A F L W V C 240 ~~AAA~C.prCAAGCATTCGCCCATCCAGG~TGGT~~GCACAATCAATMGTGAACCAGCAACACAAATGACACTTAATACT~ 84C0A ~ A T G E I E S K F L Q A L A H P G E M V G A L A A Q S I G E P A T Q M T L N T F R Y 280 E C ~ T A R G T A G T ~ T c ; T W A ~ ~ E C A C ~ T A k A C C ~ A T T A R C A ~ C A A A C ~ A C C A T C C C T C A C C A T C P A ~ A C A T A T 9G60G C T C G T A G V S S K N Y T L G V P R L N Q Z I N I A K Q V K T P S L T I Y L K P H H A R 320 GATATM;ATCG~rITGTAAAA~AAC~TATACTACACTTGCAAA~ARC TWXACCERRAWTkCTATWiTCC CTC 'IGAWCX AARATACCATCATCAG-T 1080 D M D R A X I V K S Q L E Y T T L A N V T S A T E I Y Y D P D P Q N T I I S E D 360 G C ~ T T T G T A A A T A G T T A T T T C G A R C T T C C A G A T G A A ~ T C G A T G T T C A T A G T A n : T C A C C ~ A C r r C G T A T C ~ ~ T A G A O G ? I A T O G T T A C C G A T ~ T T12 A0K0C A E A E F V N S Y F E L P D E E I D V H S M S P W L L R I E L D R G M V T D K K L T 400 A M G C C C A T A T C A C R l A A M T G T C G P T A ~ ' f i ~ T C ~ A A A ~ T A ~ A G ~ T G A T A A T G C C G A G A R A C ~ A T T C T T C G T A ~ G T A1320 TEGT~~CA M A D X T Q C V V R D F G L S L N C I F S D D N A E K L I L R I R M V E S Q E T 440 ~ T I C C C ~ ~ T G A T G A T G A T C A A T T C : C ~ G T C G T A W ~ T C A A A T A T G C T G A G ~ n ; G T T T T A C G T O G T A T C A A A G G T A 14 T4 ~0~ ~ C G A ~5 G 2r K G T D N D D D D Q F L R R I E S N M L S E M V L R G I K G I K K Y F M R T D E 480 P A R G A T K : C ~ C T G A A R A T X T G G T T T n ; G ~ T T C ~ T G G A T l K l T C 6 A T A C T G A ~ T T I Y : C C T C ~ A G A G G T A A T 6 A G C C A T C C ~ G T T G b T C A T ~ W G T1560 AL1C K I P K V T E N S G F G V R E E W I L D T D G V S L L E V M S R P D V D H T R T 520 $r A C C T C T A A W T A T C G ~ T C A T T C A G G T m P C X T A ~ C G T ~ W E E C E M m C W G W C G T T A T T T C C T T m W W C T A T G T M T T A W G T C 1680 AT T S N D I V E I I Q V L G I E A V R N A L L K E L R A V I S F D G S Y V N Y R H 560 ~~A~CGATG~ATGAC~hTCGTeGEATC~A~RTCACWGTCAn=GTATCARThGAG~TGGTCCACTCAn=AGATGTETTn:G 1800 AA~TGW~r
F1
2
L
A
I
L
A
D
V
M
T
Y
R
G
H
L
M
A
I
T
R
H
G
I
N
R
V
E
T
G
P
L
M
R
C
S
F
E
E
T
V
E
600
A T T C ~ h ~ C G C C A T G T n : T C ~ C T C A ~ T G ~ ~ T A P E T G A A A A T A T l l P T ~ A G G T C A A C T C C C A C C A ~ T A C n = G T A G T T T C G A A G T T T T C C E A192 A T0C A A I
L
M
D
A
A
M
F
S
E
T
D
D
V
K
G
V
T
E
N
I
I
L
G
Q
L
P
P
L
G
T
G
S
F
E
V
F
L
N
Q
640
-8
-g
10
GATATGA~~~C~GC~ACCAGRACCTTCCAA~~~ATCICAGATACTCCAGGTAGIYlWACCTTCCTATPCCTAWTGATCG2 T04 ~0 AC~WCWW D M I K N A H S I A L P E P S N V S Y P D T P G S Q T P S Y S Y G D G S T T P F 680 CATAAEC~ATGAn;CTCCATTGTCACCATWAA~CTTWCGTGGTGATTETCACCWTGCTATGARTn:ACCAGGTTA~RAATARATCCTATGGTPx=TAC.TTATCAA2160 H N P Y D A P L S P F N E T F R G D F S P S A M N S P G Y N A N K S Y G S S Y Q 720 T~TTC&CPCAATCACCAAcfiAT1Y:TCCAAC~AC:CATCCTA~TCCP9X:TPCACCATCTTA~CAAC'ITCTCCATC?PPATTCACC~CTPCAGCATCTCA~ACCAACTKIT 2280 Y F P Q S P T Y S P T S P S Y S P T S P S Y S P T S P S Y S P T S P S Y S P T S 760 C C A T C W A ~ C A A C C T C A C C A T C T T A ' S r C T C C A A C T T C A C C A T T P T A ~ W C ~ T T C ~ C T T C C T ~ T C A C C X T T C A C C A T C A T A C T C C C C ~ T C A C C A T C A T 24 A ~0C0A P S Y S P T S P S Y S P T S P P Y S P T S P S Y S P T S P S Y S P T S P S Y S P 800 A C C T C P v C C A T C T l l A C T C A C C A A C C ~ F X ! C C T C C T A T P C A C C A A C ~ C A T C R T A ~ A C C A A C ~ C A T C A T A ~ A C C A A C T T C ~ C A T C A T A C T C T C C ~ T P C A C C A2520 TC~AT T S F S Y S P T S P S Y S P T S F S Y S P T S P S Y S P T S P S Y S P T S P S Y 840 ~ ~ C A A C C T C T C C ~ A T ~ T C A C C A A C C T C A C C A T C T T A ~ C A R C C T C A C C A T C ~ A ~ A C C A T C R . ~ C C C A ~ ~ A ' f i c A C C A A G ~ G C C A T C A T A ~ A l : C A2640 AGTTCPI:CA S P T S P S Y S P T S P S Y S P T S P S Y S P S S P S Y S P S S P S Y S P S S P 880 TCATACTCACCATCATWCAACCTTP~AAACARATAT~TA~AACCARATAAT~AREAACATTAAGATACCATTTCCTATTIETRARTATTTAA~TATATAR 2760 AT S Y S P S S P T F T N K Y N Y Q P N N K K K 902 ~ ~ W n a A C A R A T T A A G W T A T ~ ~ P T A R T A A A ~ ~ A 2874 T A C X
FIG. 2. Nucleotide sequence of the cDNA coding for the Dictyostelium RNA polymerase I1 subunit. Nucleotides are numbered at the right in 5' to 3' direction. The deduced amino acid sequence is shown in single letter code below the nucleotide sequence and is numbered also at the right. The termination codon is in bold type and the three putative polyaclenylntion signals are underlined.
LAM ET AL.
Region I
D.d.
Mouse Yeast
?!BY#WS=%C
D@Y@A~~@EIBGc
60 909 ~ G G R E G L ~ Z T r A V I Z T ~ T G Y I Q ~ V ~ E D I M V H Y D N T T R W S L G ~ X Q P I Y G E D G M876D A A
ST
ANGGREGLIDTAVKTAETGYIQR~SLIXSM-ES~YDATVRNSINQWQLRYGEDGLAGE
*NegrBs~KDfiTELrny#l
82
~ ~ F Q M C A P L K P ~ ~ E K I 921 FFR HIEKQSLD~I G G ~ D W E 898 ~ Region X I
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
D.d. Mouse Yeast
~
S
~+T~@E%~H~&V
%
E
IE@
176
b ~ m ~ - V I ~ G D S K W L P C N L L R M I W A Q K I F & N P R ~ ; ~ S D L H P I ~ E1016 ~EL 1
D R K F L R - E V P V D G E ~ P L P W T R I U : XQNAQQTPHIDHTKPSDLTIKDIVLGVKDL 993
Region I11
B K ~ T ~ ~ @ E & ~ c
gT
D.d.
&!&I@S
Mouse Yeast
M A ' P L L L F N I H ~ S ~ C S ~ M A E E P ~ S G E A F D ~ ~ ; G E X E S K P N W A H P G E 1094 NVG~QS DAVTLPCCL;L;RSRE~TXIRVLQEY RLrTRQAFDWVfrSHTEAQPLRS SWHPGEMVGVWIQS 1071
265
'RIVK&&EY k%%iWMsAkkfWWd@dNCfl~ f SWAEFWS W&~,&E~IDVHSM--~PR~ 383 K D ? L C R L E ~ ' ~ K ~ P T A N T A I Y Y D P N P Q S T ~ A E : D Q ~ Y Y E M P D F D V A R 1210 ----SPW KLTRSAXEBTTLKSVTIASE XYYDPDPRSTVI PEDEEI IQLHFSLLDsEAEQSFDQB6'Pw
1191
Region IV
D. d. Mouse Yeast
2
~
~
@
~
@
~
476
1 , 1284
#
#
~
~
~
~
~
~
~
f
k
~ , Q ~ ~ v ; F ~ ~ c ~ ~ ~ ~ ~ SDWMY $ L 1309Q G I ~ Q I
EE$HMBKKFY$NT#~NI ,
,
50% sequence identity. The degree of homology is considerably higher if conserved amino acid changes are included in the calculation. These five conserved regions (I to V) consist of 82, 56, 228, 30, and 141 amino acids, respectively. In region I, the percentage of sequence identity is 63% with both mouse and yeast. Amino acids in region I1 show 50% identity with mouse and 55% identity with yeast. Interestingly, in regions 111-V, the percentages
of sequence identity with mouse (66, 67, and 73'70, respectively) are considerably higher than those with yeast (60,40, and 62070, respectively). Within regions I, 111, and V, there are short segments that show >80% sequence identity with either mouse or yeast. The five conserved regions are separated by segments of highly variable amino acid sequences. Computer search also revealed that the Dictyostelium sequence is homologous to the large subunit of other RNA polymerases, but to a lesser extent than the RNA polymerase I1 subunit. The most notable homologous sequence is the tandemly repeated heptapeptide at the carboxyl terminus. This tail structure is unique to the largest subunit of RNA polymerase 11, thus further confirming the identity of our cDNA clones. The CTD of the Dictyostelium polymerase subunit is composed of 24 heptapeptide repeats, with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser(Fig. 4). The same heptapeptide sequence is repeated 26 times in yeast (Allison et al. 1985) and 52 times in mouse (Ahearn et al. 1987). Variations from the consensus sequence occur to different degrees in the CTD of a number of species. In the case of Dictyostelium, there are only nine substitutions in the entire CTD domain, with one in position two, five in position four, and three in position seven, and with six of these changes being conservative. The last heptapeptide repeat is followed by a variable sequence of 14 amino acids, with three Lys residues ending at the carboxyl terminus. Gene expression during development In RNA blots, a labelled cDNA probe coding for the CTD hybridized with a single mRNA species of -6 kb (Fig. 5), which is sufficient to code for a protein of 200 kDa. The presence of an RNA polymerase I1 subunit with the unique
797
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
LAM ET AL.
FIG. 5. RNA blot and immunoblot of the largest RNA polymerase I1 subunit. Vegetative Dictyostelium cells were collected and total RNA was isolated and separated on agarose gel. In A, the RNA blot was hybridized with a 32~-labelledcDNA probe (the EcoRI fragment at the 3 ' end of the cDNA). The probe hybridized with a single RNA species of -6 kb (arrow). The positions of the 26s and 17s rRNAs are indicated on the left. Cells were also solubilized in SDS for gel electrophoresis. In B, proteins were indirectly stained with a monoclonal antibody directed against the CTD of RNA polymerase I1 largest subunit (lane a). The antibody reacted with a single protein band at 200 kDa (arrow). A similar sample reacted with preimmune serum is shown in lane b.
tail structure in Dictyostelium cells was ascertained by immunoblots. Total cellular protein derived was stained indirectly with a monoclonal antibody raised against the CTD heptapeptide sequence (Moyle et al. 1989). It reacted specifically with a protein band of -200 kDa (Fig. 5), a molecular size consistent with that of the largest subunit of RNA polymerase I1 subunit in Dictyostelium (Renart et al. 1985). To examine the expression of the largest RNA polymerase I1 subunit during development, RNA samples were isolated from cells at different developmental stages and probed with the same cDNA fragment (Fig. 6). The mRNA level increased by -60% at 3 h of development and then decreased steadily to about half the amount in vegetative cells in the next 6 h. This was followed by an accumulation of RNA transcripts between 9 and 15 h, resulting in a 3.5-fold increase in mRNA level. When cells were treated with low doses of CAMP, the general pattern of mRNA accumulation was moved forward by about 3 h. Interestingly, treating of cells with cAMP did not stimulate an increase in mRNA accumulation, but rather led to an overall reduction in mRNA level. Discussion We have cloned and sequenced cDNA coding for the carboxyl-terminal half of the largest subunit of RNA polymerase I1 from D. discoideum. The cDNA hybridized with a single mRNA species of -6 kb, sufficient to code for a protein of 200 kDa, and the amino acid sequence deduced from the cDNA sequence showed extensive sequence identity with the same RNA polymerase I1 subunit
Time of development 6. Accumulation of RNA polymerase I1 subunit mRNA during development. Vegetative cells were collected for development either in the absence of cAMP or in the presence of cAMP pulses at 7-min intervals, giving a final cAMP concentration of M. Cells were collected every 3 h during development 2 x and total RNA was isolated and separated on agarose gels. Blots were probed with a 32~-labelledcDNA probe. The same blots were also probed with actin cDNA. Autoradiograms were quantitated by densitometry and the values were normalized against the actin content. The relative levels of mRNA were estimated for samples collected at different time points, with stippled bars for the nonpulsed control and the striped bars for the CAMP-pulsed sample. Values represent the average of two experiments. FIG.
in many different species. Sequence analysis reveals that the RNA polymerase subunits are a group of highly conserved proteins. Sequences of the largest RNA polymerase I1 subunit of Saccharomyces cerevisiae (Allison et al. 1985), Caenorhabditis elegans (Bird and Riddle 1989), Drosophila melanogaster (Jokerst et al. 1989), and the mouse (Ahearn et al. 1987) show that almost 40% of the amino acid residues are invariant. Interestingly, many of the conditional mutations of RNA polymerase I1 affect these invariant amino acid residues (Scafe et al. 1990), suggesting that the conserved segments of the protein may have important structural or functional roles. A remarkable feature of the eukaryotic RNA polymerase I1 is the presence of a heptapeptide repeat in the CTD of its largest subunit. The Dictyostelium subunit has a CTD consisting of 24 repeats of the consensus sequence: Tyr-SerPro-Thr-Ser-Pro-Ser. This consensus sequence is repeated 52 times in mouse (Corden et al. 1985) and hamster (Allison et a1 1988), 42-44 times in Drosophila (Allison et al. 1988; Zehring et al. 1988), 32 times in C. elegans (Bird and Riddle 1989), 26-27 times in yeast (Allison et al. 1985), and 17 times in Plasmodium falciparum (Li et al. 1989). The length of the CTD seems to be related to the complexity of the organism. The amino acids in positions one (Tyr), three (Pro), and six (Pro) are nearly invariant, while the amino acid at position seven shows a high degree of divergence. The Dictyostelium CTD is highly conserved, with 18 exact matches out of 24 repeats. In comparison, yeast has 17 exact matches out of 26, while Drosophila has the most divergent
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
798
BIOCHEM. CELL BIOL. VOL. 70,
CTD, with only 2 of the 42-44 repeats matching the consensus sequence. The CTD of RNA polymerase I1 is likely to be fully exposed to the solvent and project out from the globular fold of the rest of the polypeptide, since all the amino acid side chains, excepting that of proline, are hydrophilic. The repeated pattern of this domain also suggests a regular, flexible structure. Genetic studies show that deletions of all or most of the repeats in the yeast or mouse CTD are lethal (Allison et al. 1988; Zehring et al. 1988). These results imply that the CTD has an essential role in transcription. However, the precise function of the CTD is still unknown. It is possible that the extended CTD may be involved in interaction with certain transcriptional factors at a distance (Corden and Ingles 1992), thus facilitating the recruitment of trans-acting factors to the transcription initiation complex. However, the nature of such target proteins remains to be identified. Another possibility is that the CTD may participate in transcription initiation through direct interaction with DNA (Suzuki 1990). In both models, the role of the CTD in transcriptional regulation may be mediated by phosphorylation and dephosphorylation reactions, since the CTD is known to be heavily phosphorylated in vivo (Cadena and Dahmus 1987). The expression of RNA polymerase I1 is essential for growth and development and is expected to behave like a housekeeping gene. However, our results show that the expression of the largest subunit of RNA polymerase I1 is under developmental regulation. During the first 3 h of Dictyostelium development, its mRNA level rose slightly and then decreased steadily for the next 6 h. This corresponded to a period when the number of mRNA species does not change significantly (Blumberg and Lodish 1980). Although the expression of some genes are induced, the number of such genes is relatively low (Alton and Lodish 1977; Mangiarotti et al. 1983). The metabolic activity of cells during this initial period of starvation is apparently lower and a decrease in the overall synthesis of glycoprotein has been observed (Lam and Siu 1981). By contrast, about 2500 new species of mRNA are synthesized in postaggregation cells (Blumberg and Lodish 1980). The transcription of this large number of developmentally regulated genes is preceded by an increase in the mRNA level of the largest subunit of RNA polymerase 11, which begins at the late-aggregation stage. A close temporal correlation therefore exists between the increase in RNA polymerase I1 expression and the elevated transcriptional activities in the postaggregation stages of development. Many genes expressed during the aggregation and postaggregation stages are under the control of CAMP.A number of early genes are sensitive to pulses of low concentrations of CAMP,while the postaggregation stage genes require a constant level of much higher concentration of cAMP for stimulation (for a review, see Firtel 1991). However, cAMP does not appear to have a major effect on the mRNA level of the largest subunit of RNA polymerase 11, except that the pattern of mRNA levels was shifted 3 h forward. This is consistent with the fact that exogenously added cAMP can accelerate the developmental program by 3-4 h. cAMP also caused an overall reduction in the mRNA level of the RNA polymerase I1 subunit. Whether this is due to a decrease in mRNA stability or a general reduction in the transcription of housekeeping genes is not clear. This unique
1992
pattern of expression of the largest subunit of RNA polymerase I1 suggests a regulatory mechanism differing from those involved in the expression of stage-specific genes. Further analysis of its gene and promoter region will be required to understand the regulation of its expression. Acknowledgements We thank Dr. Jim Ingles for his advice and his generous gift of monoclonal antibody directed against the CTD of the largest subunit of RNA polymerase 11. This work was supported by an operating grant from the Medical Research Council of Canada and L. Chan was supported by a summer studentship from the Life Sciences Committee, University of Toronto. Ahearn, J.M., Bartolomei, MS., West, M.L., Cisek, L.J., and Corden, J.L. 1987. Cloning and sequence analysis of the mouse genomic locus encoding the largest subunit of RNA polymerase 11. J. Biol. Chem. 262: 10 695 - 10 705. Allison, L.A., Moyle, M., Shales, M., and Ingles, C.J. 1985. Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases. Cell, 42: 599-610. Allison, L.A., Wong, J.K., Fitzpatrick, D., Moyle, M., and Ingles, C.J. 1988. The C-terminal domain of the largest subunit of RNA polymerase I1 of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: a conserved structure with an essential function. Mol. Cell. Biol. 8: 321-329. Alton, T.H., and Lodish, H.F. 1977. Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum. Dev. Biol. 60: 180-206. Bartolomei, M.S., Halden, N.F., Cullen, C.R., and Corden, J .L. 1988. Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase 11. Mol. Cell. Biol. 8: 330-339. Bird, D.M., and Riddle, D.L. 1989. Molecular cloning and sequencing of ama-1, the gene encoding the largest subunit of Caenorhabditis elegans RNA polymerase 11. Mol. Cell. Biol. 9: 4119-4130.
Blumberg, D.D., and Lodish, H.F. 1980. Changes in the messenger RNA population during differentiation of Dictyostelium discoideum. Dev. Biol. 78: 285-300. Cadena, D.L., and Dahmus, M.E. 1987. Messenger RNA synthesis in mammalian cells is catalyzed by the phosphorylation form of RNA polymerase 11. J. Biol. Chem. 262: 12 468 - 12 474. Cho, K.W.Y.. Khalili, K., Zandomeni, R., and Weinmann, R. 1985. The gene encoding the largest subunit of human RNA polymerase 11. J. Biol. Chem. 260: 15 204 - 15 210. Cocucci, S., and Sussman, M. 1970. RNA in cytoplasmic and nuclear fractions of cellular slime mold amebas. J. Cell Biol. 45: 399-407. Corden, J.L., and Ingles, C.J. 1992. The carboxyl-terminaldomain of the largest subunit of eucaryotic RNA polymerase 11. In Transcriptional regulation. Edited by K.R. Yamamoto and S. McKnight. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. In press. Corden, J.L., Cadena, D.L., Aheam, J.M., and Dahmus, M.E. 1985. A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase 11. Proc. Natl. Acad. Sci. U.S.A. 82: 7934-7938. Dente, L., Cesareni, G., and Cortese, R. 1983. pEMBL: a new family of single stranded plasmids Nucleic Acids Res. 11: 1645-1655.
Evers, R., Hammer, A., Kock, J., Jess, W., Borst, P., Memet, S., and Cornelissen, A. W. 1989. Trypanosoma brucei contains two RNA polymerase I1 largest subunit genes with an altered C-terminal domain. Cell, 56: 585-597.
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only.
LAM ET AL.
Firtel, R.A. 1991. Signal transduction pathways controlling multicellular development in Dictyostelium. Trends Genet. 7 : 381-388. Henikoff, S. 1984. Unidirectional digestion with exonuclease I11 creates targeted breakpoints for DNA sequencing. Gene, 28: 351-359. Ingles, C. J., Biggs, J., Wong, J.K.-C., Weeks, J.R., and Greenleaf, A.L. 1983. Identification of a structural gene for an RNA polymerase I1 polypeptide in Drosophila melanogaster and marnmalian species. Proc. Natl. Acad. Sci. U.S.A. 80: 8896-3400. Jokerst, R.S., Weeks, J.R., Zehring, W.A., and Greenleaf, A.L. 1989. Analysis of the gene encoding the largest subunit of RNA polymerase I1 in Drosophila. Mol. Gen. Genet. 215: 266-275. Kimmel, A., and Firtel, R.A. 1983. Sequence organization in Dictyostelium: unique structure at the 5'-ends of protein coding genes. Nucleic Acids Res. 11: 541-552. Laemmli, V.K. 1970. Cleavage of structural proteins during assembly of the bacteriophage T4. Nature (London), 227: 680-685. Lam, T.Y., and Siu, C.-H. 1981. Synthesis of stage-specific glycoproteins in Dictyostelium discoideum. Dev. Biol. 83: 127-137. Li, W.B., Bzik, D.J., Gu, H., Tanaka, M., Fox, B.A., and Inselburg, J. 1989. An enlarged largest subunit of Plasmodium falc@arum RNA polymerase I1 defines conserved and variable RNA polymerase domains. Nucleic Acids Res. 17: 9621-9636. Loomis, W.F. 1975. Dictyostelium discoideum: a developmental system. Academic Press, Inc., New York. pp. 1-85. Ma, P.C.-C., and Siu, C.-H. 1990. A pharmacologically distinct cyclic AMP receptor is responsible for the regulation of gp80 expression in Dictyostelium discoideum. Mol. Cell. Biol. 7 : 3297-3306. Mangiarotti, G., Bozzaro, S., Landfear, S., and Lodish, H.F. 1983. Cell-cell contact, CAMP, and gene expression during development of Dictyostelium discoideum. Curr. Top. Dev. Biol. 18: 117-154. Mann, S.K.O., and Firtel, R.A. 1989. Two-phase regulatory pathway controls CAMPreceptor-mediated expression of early genes in Dictyostelium. Proc. Natl. Acad. Sci. U.S.A. 86: 1924- 1928. Memet, S., Gouy, M., Marck, C., Sentenac, A., and Buhler, J.-M. 1988. The gene coding for the largest subunit of yeast RNA polymerase A. J. Biol. Chem. 263: 2830-2839. Moyle, M., Lee, J.S., Anderson, W.F., and Ingles, C.J. 1989. The C-terminal domain of the largest subunit of RNA polymerase I1 and transcription initiation. Mol. Cell. Biol. 9: 5750-5753. Pong, S., and Loomis, W.F. 1973. Multiple nuclear RNA polyrnerases during development of Dictyostelium discoideum. J. Biol. Chem. 248: 3933-3939. Proudfoot, N.J., and Brownlee, G.G. 1976. 3' non-coding region
799
sequences in eukaryotic messenger RNA. Nature (London), 263: 21 1-214. Renart, M.F., Sastre, L., Diaz, V., and Sebastian, J. 1985. Purification and subunit structure of RNA polymerase I and I1 from Dictyostelium discoideum vegetative cells. Mol. Cell. Biochem. 66: 21-29. Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467. Sawadogo, M., and Sentenac, A. 1990. RNA polymerase B (11) and general transcription factors. Annu. Rev. Biochem. 59: 71 1-754. Scafe, C., Nonet, M., and Young, R.A. 1990. RNA polymerase I1 mutants defective in transcription of a subset of genes. Mol. Cell. Biol. 10: 1010-1016. Searles, L.L., Jokerst, R.S., Bingham, P.M., Voekler, R.A., and Greenleaf, A.L. 1982. Molecular cloning of sequences from a Drosophila RNA polymerase I1 locus by P element transponson tagging. Cell, 31: 585-592. Smith, J.L., Levin, J.R., Ingles, C.J., and Agabian, N. 1989. In trypanosomes the homolog of the largest subunit of RNA polymerase I1 is encoded by two genes and has a highly unusual C-terminal domain structure. Cell, 56: 815-827. Sussman, M. 1966. Biochemical and genetic methods in the study of cellular slime mold development. Methods Cell Physiol. 2: 397-409. Suzuki, M. 1990. The heptad repeat in the largest subunit of RNA polymerase I1 binds by intercalating into DNA. Nature (London), 344: 562-565. Towbin, H., Staehelin, T., and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354. Warrick, H.M., and Spudich, J.A. 1988. Codon preference in Dictyostelium discoideum. Nucleic Acids Res. 16: 6617-6635. Wong, L.M., and Siu, C.-H. 1986. Cloning of cDNA for the contact site A glycoprotein of Dictyostelium discoideum. Proc. Natl. Acad. Sci. U.S.A. 83: 4248-4252. Woychik, N.A., and Young, R.A. 1990. RNA polymerase 11: subunit structure and function. Trends Biochem. Sci. 15: 347-350. Young, R.A., and Davis, R.W. 1983. Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. U.S.A. 80: 1194-1 198. Zehring, W.A., Lee, J.M., Weeks, J.R., Jokerst, R.S., and Greenleaf, A.L. 1988. the C-terminal repeat domain of RNA polymerase I1 largest subunit is essential in vivo but is not required for accurate transcription in vitro. Proc. Natl. Acad. Sci. U.S.A. 85: 3698-3702.
