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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 LITERATURE CITED BAUTZ,E. K. F., T. KASAI,E. REILLYand F.A. BAUTZ,1966 Gene specific mRNA. 11. Regulation of mRNA synthesis in E. coli after infection with bacteriophage T4. Proc. Natl. Acad. Sci. USA 55: 1081-1088. K. L., T. ISHII and A. TSUGITA,1978 Complete primary structure of the small outer BIJLENGA, capsid (SOC)protein of bacteriophage T4. J. Mol. Biol. 120 249-263. BRODY,E. N., D. RABUSSAY and D. H. HALL,1983 Replication of transcription of pre-replicative E. M. KUTTER,G. MOSIG genes.pp. 174-183. In: Bacteriophage T4, Edited by C. K. MATHEWS, and P. B. BERGET.American Society for Microbiology, Washington, D. C. CHRISTENSEN, A. C. and E. T . YOUNG,1982 T 4 late transcripts are initiated near a conserved DNA sequence. Nature 299 369-37 1. CHRISTENSEN, A. C., and E. T. YOUNG,1983 Characterization of T 4 transcripts. pp. 184-188. In: Bacteriophage T4, Edited by C. K. MATHEWS,E. M. KUTTER,G. MOSIG and P. B. BERGET. American Society for Mircrobiology, Washington, D. C. and L. GOLD,1984 Translational control mediated CHRISTENSEN, A. C., E. T. YOUNG,G. STORMO by transcriptional control: the T 4 lysozyme gene. J. Virol. In press. FORREST, G. L. and D. J. CUMMINGS, 1971 Head proteins from T-even bacteriophages. 11. 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