Download regulation of a bacteriophage t4 late gene, soc, which

Copyright 0 1984 by the Genetics Society of America
REGULATION OF A BACTERIOPHAGE T4 LATE GENE,
WHICH MAPS IN AN EARLY REGION
SOC,
PAUL M. MACDONALD,* ELIZABETH KUTTERt AND GISELA MOSIG*
* Department of Moleculnr Biology, Vnnderbilt University, Nashville, Tennessee 37235; and
The Evergreen
State College, Olyinpia, Washington 9 8 5 0 5
Manuscript received July 30, 1983
Revised copy accepted September 20, 1983
ABSTRACT
We have sequenced and analyzed the expression of an early region of the
bacteriophage T 4 genome that surprisingly contains a late gene, SOC. soc is oriented
in the same direction as early genes, like the T 4 lysozyme gene. Northern
hybridization of early and late T 4 RNA, using cloned T 4 restriction fragments
as probes, identified two long early transcripts and a short late transcript, all
containing the soc-coding sequence. Thus, soc is transcribed both early and late.
It is, however, translated only late. The inhibition of soc translation from the
long early transcripts can be explained by formation of a hairpin in the RNA
that sequesters the soc ribosome-binding site. The transcript initiated at the late
promoter cannot form this hairpin and is, therefore, translated.
ACTERIOPHAGE T 4 development depends
precise temporal control of
B
gene expression (for a detailed review, see RABUSSAY
1982, 1983). Most
bacteriophage T 4 genes coding for enzymes required in T 4 DNA metabolism
on
are expressed early, either immediately after infection or slightly delayed (O’FARRELL and GOLD1973; BRODY,RABUSSAY
and HALL1983). Genes involved in
DNA packaging are expressed late. T h e shift from early to late gene transcription
has at least two requirements. (1) Early T 4 proteins associate with the E. coli
RNA polymerase and alter its specificity such that only late T 4 promoters are
recognized (PULITZER
and GEIDUSCHEK
1970; RABUSSAY
1983). (2) T h e template
must become competent either by replication or by exposure to certain nucleases
under DNA ligase-deficient conditions (RIVA,CASCINO
and GEIDUSCHEK
1970a,
b). In general, clusters of early and late genes are well separated in the genome
and are transcribed from opposite strands (GUHAet al. 197 1).
Capsids of deletion mutant del(39-56)12 phage, isolated by HOMYKand WEIL
(1974), lack the SOCprotein, suggesting that soc or a gene controlling expression
of soc is missing in the mutant (ISHIIand YANAGIDA1977). T h e small outer capsid
protein (gpSoc) is a nonessential exterior component of the T 4 head (FORREST
and CUMMINGS
1971; ISHII and YANAGIDA 1975). It is synthesized only late in
infection (ISHIIand YANAGIDA1975; MOSIG et al. 1983). T h e absence of SOC
protein, which is normally present in T 4 heads at the same copy number as the
major head protein (gp23*), renders the head less resistant to inactivation by
alkaline conditions (ISHII and YANAGIDA 1977). T h e SOC protein has been
1978).
sequenced (BIJLENGA,
ISHIIand TSUGITA
We have recently cloned and sequenced part of the region of the T 4 genome
Genetics 106: 17-27 January, 1984.
18
P. M. MACDONALD, E. KUTTER AND G. MOSIG
deleted in del(39-56)12 (MACDONALDand MOSIG 1983 and unpublished; MACDONALD et al. 1983). One segment of the DNA sequence deleted in del(39-56)12
(see Figure 7 of MACDONALDand MOSIG 1983) contains an open reading frame
that could code for an 80-amino acid protein (Figure 1). We conclude that this
1
Ala S e r Thr
Met A l a S e r Thr
Phe Arg Lys Glu Asp Val end
TTC AGA AAA GAA GAT GTA
TAA_ATAATCATGTAATTTAAATAAAGGAGAATTAC ATG GCT AGT
ACT
__
__
l a t e promoter
RsaI
5
10
15
Arg Gly Tyr V a l Asn I l e Lys Thr Phe Glu Gln Lys Leu Asp Gly
Arg Gly Tyr Val Asn I l e Lys Thr Phe Glu Gln Lys Leu Asp Gly
CGC GGT TAT GTT AAT ATC AAA ACA TTT GAG CAG AAA TTA GAT GGA
25
30
Glu Gly Lys Glu I l e Ser Val A l a Phe Pro Leu
Glu Gly Lys Glu I l e S e r V a l A l a Phe Pro Leu
GAA GGA AAG GAA ATT TCT GTA GCT TTC CCT CTT
20
Asn Lys Lys I l e
Asn Lys Lys I l e
AAT AAG AAA ATT
35
39
Tyr S e r Asp Val H i s Glu Gly Lys
Tyr S e r Asp Val H i s
TAT TCT GAC GTT CAC
40
45
50
55
Glu Phe H i s Lys I l e S e r Gly A l a Thr Tyr Gln Thr Phe P r o S e r Glu Lys A l a Ala
Lys I l e S e r Gly A l a H i s Tyr Gln Thr Phe Pro S e r Glu Lys A l a A l a
AAA ATT TCT GGC GCT CAT TAC CAG ACA TTC CCT TCA GAA AAA GCA GCA
Hae I1
_.-
60
65
70
Tyr S e r Thr V a l Tyr Glu Glu Asn Gln Arg Thr Glu Trp I l e A l a A l a Asn Glu Asp
Tyr S e r Thr V a l Tyr Glu Glu Asn Gln Arg Thr Glu Trp I l e A l a A l a Asn Glu Asp
TAT TCT ACA GTA TAT GAA GAA AAT CAA CGT ACT GAA TGG ATT GCT GCA AAT GAA GAT
RsaI
75
80
Leu Trp Lys Gly Val Thr
Leu Trp Lys V a l Thr Gly end end
TTG TGG AAA GTA ACT GGT TAA TAA CTCAAGGACTCCTTCGGAGTCCTTTTTCATTTAAA
TGGTTTACTTTCCAAAATGAGTATGGTATAATAG
-35
-10
FIGURE1.-DNA sequence of the gene SOC. Both strands of the DNA from this region were
sequenced by the partial chemical degradation method (MAXAMand GILBERT1980), using various
and
DNA restriction fragments from a cloned 0.85-kb EcoRI fragment (pMAC21, MACDONALD
MOSIG 1983). Restriction fragments were 5’-end labeled with polynucleotide kinase or 3’-end labeled
by filling in staggered ends with E. coli polymerase I (Klenow fragment). The deduced amino acid
sequence of sac and of the carboxy-terminal region of an early protein in the ori region (P. MACWNALD
and G. MOSIG,mpublished results) are indicated, aligned with the amino acid sequence of the
purified SOCprotein. The T 4 late promoter sequence upstream from sac (see text) is underlined. The
-35and -10 regions of an early promoter located downstream from sac, PE14.98 (see Figure 4), are
also underlined. This promoter was identified and mapped by in vitro transcription experiments (P.
MACWNALD,unpublished results). Transcripts initiated at P~14.98in vivo are also detected by the
Northern hybridization experiments (Figure 3).
19
REGULATION OF T 4 GENE Soc
open reading frame is the coding sequence of soc because the amino acid sequence
predicted by the DNA sequence closely resembles the sequence of purified Soc
protein (BIJLENGA,ISHII and TSUGITA
1978; see Figure 1). Analyses of T2/T4
heteroduplexes are consistent with this map position of SOC. T2 particles lack the
Soc protein (ISHII and YANAGIDA1975)and a single-stranded loop approximately
15 kb in the clockwise direction from the rllA/Bjunction is formed in T 4 DNA
hybridized to T 2 DNA (KIM and DAVID" 1974).
There are four minor differences between the amino acid sequence of the
purified SOCprotein and that predicted by the DNA sequence (Figure 1). (1)
The DNA sequence predicts an additional N-terminal amino acid, methionine.
This residue is probably cleaved posttranslationally. (2) A six-amino acid section
in the middle of the published protein sequence is not predicted by the DNA
sequence. A representative DNA sequencing ladder of this region is shown in
Figure 2a. (3) T h e threonine at position 45 in the protein sequence reads as a
G
A
T
C
2' 5'
r,
G
A
T
C
Lr
G&
MI
Thr
T A Ochre
flhrJ
A
"1
J
G C lyr46
0
T A
b
FIGURE2.-Autoradiograms of DNA-sequencing gels showing the positions of disagreement with
the soc protein sequence. Lanes G , A, T and C are products of the G , G + A. C + T and C partial
chemical degradation-sequencing reactions. respectively. For both (a) and (b) the antimessage DNA
strand was used for sequencing. Numbering of amino acids is the same as for Figure 1. a. T h e DNA
sequence read from this portion of the gel includes the site where six additional amino acids are
located in the published protein sequence. T h e histidine codon in the DNA sequence corresponding
to a threonine residue in the protein sequence is also shown. b, T h e DNA sequence of the end of the
soc gene, showing the codons of the Cterminal tripeptide. T h e published terminal tripeptide of the
Soc protein is indicated in brackets.
20
P. M. MACDONALD, E. KUTTER AND G. MOSIG
1
2
FIGURE3.-Northern blot analysis of RNA from the soc region. RNA was isolated by boiling
infected cells in SDS (HAGENand YOUNG1978). centrifuging through cesium chloride (GuSIN.
CRKVENJAKOV
and BYUS 1974; ULLRICH
rt al. 1977) and digesting with RNascfree DNase (Miles
Laboratories) in the presence of 4 mM vanadyl-ribonucleosides (Bethesda Research Laboratories).
RNA (20 pgflane) was denatured with glyoxal and dimethylsulfoxide and electrophoresed into a
1.1 % agarose gel (MCMASTERand CARMICHAEL
1977), transferred to nitrocellulose (THOMAS
1980)
and hybridized to '*P-labeled probes. RsuI restriction fragments 1 and 2 in Figure 4, isolated after
digestion of a cloned EmRI fragment (pMAC21, MACDONALDand MOSIG 1983) were labeled with
T4 DNA polymerase by replacement synthesis (OFARRELL,
KWITFB and NAKANISHI
1980). purified
by gel electrophoresis and used as probes. Panel 1. Autoradiograph of probe 1 (see Figure 4)
hybridized to a Northern blot of early RNA (isolated 6 min after infection at 30") from E. coli B
REGULATION OF T4 GENE SOC
21
histidine in the DNA sequence, as seen in Figure 2a. (4) T h e three-amino acids
at the carboxy terminus appear in a different order: -Val-Thr-Gly is predicted
by the DNA sequence, and -Gly-Val-Thr is the published protein sequence. A
DNA-sequencing ladder of this region of the gene is shown in Figure 2b. T w o
independently derived clones gave the same DNA sequence of the entire gene.
We do not know the reyons for the inconsistencies between the DNA and
protein data. Differences in the T 4 strains used may be at least partially responsible. In addition, some portions of the protein were difficult to sequence because
1978).
of hydrophobicity (BIJLENGA, ISHIIand TSUGITA
T h e position and orientation of the soc gene are unusual for a late gene. soc
maps between known early genes (see KUTTER and RUGER 1983), e.g., dam
(HATTMAN1983) and mod (HORVITZ
1974). T h e restriction mapping (MACDONALD and MOSIG 1983) and sequence data (Figure 1) together with the Northern
blots (Figure 3) show that soc is oriented in the early, counterclockwise direction.
T h e Northern blots in Figure 3 also show that the soc sequence is transcribed
early from two promoters located about 750 and 1250 base pairs (bp) upstream,
as well as late from a late promoter-like sequence immediately upstream from
the soc-coding sequence (see Figure 1). T h e 1050- and 1550-base early transcripts
(marked with closed circles in Figure 3) found both in del(39-56)4 and in the
3 3 - 5 7 mutant were detected with the RsaI probe 2 but not with the RsaI probe
1 shown in Figure 4. Both transcripts were also detected by hybridizing with a
probe labeled only in the strand complementary to early RNA (data not shown).
Therefore, we conclude that these transcripts were terminated within the DNA
segment corresponding to probe 2 in Figure 4. This segment contains a sequence
resembling factor-independent terminators (Figure 5), identified as t15.02 in
Figure 4. This sequence is likely responsible for the termination of transcription.
The length of the 1050- and 1550-base early transcripts indicates that they are
initiated at PEl6.08 and PE16.57,respectively. Thus, both transcripts include all
of the soc-coding sequence.
A short (approximately 300-base) transcript (marked with a closed square in
Figure 3), detected with probe 2 but not with probe 1, was present only in the,
late RNA samples (Figure 3, panel 2, lane B). The size of this late RNA and its
hybridization pattern are consistent with initiation of transcription from a T 4
late promoter sequence (PL15.32)and termination at t15.02. CHRISTENSEN
and
YOUNG(1982) have deduced a T 4 late promoter consensus sequence, TATAAATACTATT,
based on S1 mapping of four T 4 late transcripts. T h e underlined
infected with (A) dd(39-56)4 or ( C ) 33-55-, and to (B) late RNA (20 min, 30") from E. coli B infected
with dd(39-56)4. Panel 2, Hybridization of probe 2 (see Figure 4) to the same RNA species as in
panel 1 . de1(39-56)4(HOMYK
and WEIL1974) is a T4 mutant with a deletion that ends approximately
0.4 kb downstream from Soc. 33-55- is a double amber mutant (N134-BL292) that does not make
late transcripts in E. coli B (PULITZER
and GEIDUSCHEK
1970). In addition to the 1050- and 1550base early soc transcripts and the approximately 300-base late soc transcript, three early transcripts
initiated downstream from soc were found. One transcript of 1150 bases in de1(39-56)4 and two
transcripts of 1350- and 2 150-bases in the 33-55- mutant were detected with both probes 1 and 2
and not with any other probe from the region upstream of t15.02 (data not shown). All of these
transcripts were probably initiated at PE14.98shown in Figure 4, an early promoter identified and
mapped by i t ? nitro transcription experiments (P. MACDONALD,unpublished results).
22
P. M.
MACDONALD, E.
KUTTER AND G. MOSIG
1
SOC
14 8
Map
15.0
1
I
RsaI
probes
I
I
15.2
I
I
RsaI
I
I
Rsal
2
I
I
HaeII
+
-1350 and 2150 bases PE14.98
*
t15.02
I
I
I-
Transcripts
I
RsoI
300 bases
I
PI 15.32
1050 and 1550 bases
/mh
P ~ 1 6 . 0 8 P ~ 1 57
6
FIGURE4.-The soc region of the bacteriophage T 4 genome. The map coordinates represent the
distance (in kilobases) clockwise from the rllA/rZZBjunction. The segment shown is part of the 0.85kb EroRI fragment (coordinates 14.77-15.62) (MACDONALD
and MOSIG1983). All RsaI restriction
sites are indicated. Probes used for the Northern hybridization experiments (Figure 3) are the RsaI
fragments labeled 1 and 2. The Hoe11 site allows alignment with the T4 restriction map (MACDONALD
and MOSIG 1983; KUTTERand RUGER1983). The transcripts that were identified as described in the
Figure 3 legend are shown.
bases are present in all four promoters. A similar sequence, TATAAATAATCAT, is found beginning 37 bp upstream from the SOC AUG codon (Figure 1).
All of the invariant bases and ten of 13 bases of the overall consensus sequence
are conserved. CHRISTENSEN
and YOUNG (1982, 1983) also noted that this
consensus sequence was not found anywhere in the T 4 DNA that had been
and YOUNG 1982,
sequenced, except in front of six late genes (CHRISTENSEN
1983; OLIVERand CROWTHER
1981; OWENet al. 1983), and in front of gene 32
(KRISCH and ALLET1982),which is expressed both early and late. We conclude,
therefore, that a T 4 late promoter (PL15.32 in Figure 4) is located directly
upstream from SOC, and that the approximately 300-base transcript (Figure 3)
initiated from this promoter is responsible for the late expression of SOC. The size
and location of this short transcript from the SOC region are consistent with
initiation at that late promoter-like sequence, transcription through SOC, and
termination at t15.02 (see Figures 4 and 5), where the two early transcripts
mentioned before also terminate. The orientation of SOC is also consistent with
transcription in this direction, counterclockwise on the T 4 map.
What inhibits translation of the early SOC message? Several factors are likely to
contribute. CHRISTENSEN
et al. (1984) have suggested an elegant mechanism for
selective translation of the T 4 lysozyme message. This gene is transcribed in the
“early”direction both early and late but is translated only late (BAUTZet al. 1966;
KASAIand BAUTZ1969,JAYARAMAN and GOLDBERG
1970). The DNA sequence
of the lysozyme gene determined by OWENet al. (1983) suggests that a palindrome in the long early transcript can form a stem-loop structure that sequesters
the ribosome-binding site for lysozyme message. A late promoter that is closer
to the coding sequence directs transcription of a message that lacks part of the
et al. 1983). Consequently, the late transcript cannot
palindrome (CHRISTENSEN
form the stem-loop structure, and the ribosome-binding site is exposed. ISERENTANT and FIERS(1980) have suggested that mRNA ribosome-binding site secondary structure influences efficiency of translation initiation.
This model applies equally well to SOC. The two long early transcripts initiated
upstream from SOC should be able to form the palindromic stem-loop structure
23
REGULATION OF T4 GENE SOC
A
A,
A
A
U
G
A
A
U*G
A
A
U
G
A
G
A
A
6,
G
A
A
A
U G
U*A
U
U*A
A* U
A*U
U* A
U A
A
A
A U
5 ' A U
A
C
G C U A3'
T
C
G
T
C O G
C * G
T * A
C
A
G
G
A
*
*
*
*
*
G
T
C
C
T
5 ' C T C A . T T T T C A T T T 3'
FIGURE5.-A and B, Secondary structures of RNA at the soc translation initiation site. The AUG
codon and the sequence corresponding to the T 4 late promoter are boxed. A, Early transcripts
initiated from both P ~ 1 6 . 0 8and PE16.57 can form a stem-loop structure that includes most of the
ribosome-binding site. A stem-loop formed in the DNA would probably be smaller (no G U bp) but
would still include part of the T 4 late promoter sequence. B, Late transcripts initiated from the T 4
late promoter sequence ( P ~ 1 5 . 3 2are
) less likely to form a stem-loop structure because they lack most
of the stem if they are initiated 3 bp downstream from the late promoter sequence (see CHRISTENSEN
and YOUNG 1982). C, The hairpin followed by a run of thymidines that is presumably responsible
for the observed termination downstream from soc (Figure 3) of the two long early transcripts initiated
at PE16.08and PE16.57 and the short late transcript initiated at P ~ 1 5 . 3 2 .
shown in Figure 5A. Here the ribosome-binding site is sequestered, presumably
preventing initiation of translation. Note that base pairing of the AUG start
codon within the palindromic sequence is facilitated by the divergence of two
bases from the T 4 late promoter consensus sequence. On the other hand, the
24
P. M. MACDONALD, E. KUTTER AND G. MOSIG
short late transcripts (Figure 3) initiated several bases downstream from the late
promoter-like sequence would retain only a portion of the palindrome. A stemloop structure in the mRNA is then less likely to form, and translation from the
now exposed ribosome-binding site can be initiated. Interestingly, the four bases
following the soc start codon are GCTA, a sequence that frequently follows the
start codon in the messages for abundant proteins (GOLDet al. 1981), such as
gpsoc.
Late expression of soc is greatly enhanced in infections of a ts gyrB E. coli
mutant (ORRet al. 1979) by a T 4 topoisomerase (397 mutant at 42" (MOSIGet
al. (1983). This suggests that initiation of transcription from the late promoter
is somehow depressed under normal conditions and that this depression is
relieved when both host gyrase and T 4 topoisomerase are defective. This effect
may involve the same palindromic sequence in the DNA. It is possible that a
stem-loop structure sequestering the late promoter can be formed in vivo in the
DNA as a consequence of torsional stress combined with the activity of DNA
unwinding proteins. If the stem-loop is formed less efficiently as a result of the
host and phage topoisomerase mutations, the promoter is more accessible and
the gene appears overexpressed. Other promoters that have apparent low activity
under ordinary conditions are located in similar positions of palindromic sequences. Examples are the putative transposase promoter of IS4 (KLAER et al.
198l), the lit promoter of phage X (LANDSMANN, KROGERand HOBOM1982) and
the Diphtheria tox promoter (KACZOREKet al. 1983). Synthesis of the lit transcript,
et al. 1982), is greatly enhanced,
which codes for the Rex B protein (LANDSMANN
1973). DNA
in cis only, by concomitant DNA replication (HAYESand SZYBALSKI
replication through this region could eliminate the hairpin or prevent it from
forming. It will be interesting to see whether activities of such promoters are
generally influenced by torsional stress in the DNA.
Even in this nonessential region of the genome there is efficient use of the
informational capacity of the DNA. Upstream from soc is an open reading frame
for an early protein (P. MACDONALDand G . MOSIG, unpublished results). The
last amino acid codon and the stop codon of that sequence overlap with the soc
late promoter sequence (Figure 1). Furthermore, there is an early promoter
located just downstream from soc (Figure 1).
The location and the orientation of both soc and e are contrary to the majority
of described late genes. Are these genes truly exceptional or are they examples
of a larger class of still unidentified genes that do not adhere to our preconceived
notions of T 4 gene arrangements? Interestingly, initiation of RNA synthesis in
the early direction within regions coding for late genes has recently been reported
(GEIDUSCHEK,
ELLIOTand KASSAVETIS 1983; CHRISTENSEN
and YOUNG 1983).
Perhaps "misplaced" genes in the T 4 genome are more common than had been
thought.
This work was supported by Public Health Service grant GM 13221, Biomedical Research grant
RR07201 from the National Institutes of Health, and Natural Science Fund of Vanderbilt, to G . M.,
and a National Science Foundation grant PCM 7905626 to E. K. P. M. was supported by National
Institutes of Health Cellular/Molecular Biology Graduate Training grant T 3 2 GM073 19. We thank
A. CHRISTENSEN
for sending their manuscript prior to publication.
REGULATION OF T4 GENE SOC
25
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Corresponding editor: J. W. DRAKE