THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264, No. 24, Issue of August 25, pp. 14432-14439, 1989 Printed in U. S. A.

Transcript Analyses of the UUSX-40-41 Region of Bacteriophage T4 CHANGES IN THE RNA AS INFECTION PROCEEDS*

(Received for publication, January 19, 1989)

Deborah M. Hinton From the Section on Nucleic Acid Biochemistry, Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

The bacteriophage T4 genes UUSX (recombination protein), 40 (stimulates head formation), and 41 (DNA replication protein, part of the primase-helicase) are located together on the T4 genome (5’ + 3‘ uusX-40- 41). Previous analyses have indicated that all three proteins are expressed within 5 min after infection and that the level of 41 protein is less than that of UVSX. The mapping of transcripts from this region (reported here) shows that this expression arises from polycis- tronic messages detected between 2-4 min after infec- tion, a time when phage-encoded factors are beginning to alter the host transcriptional apparatus. Major RNA 5’ ends, 900 and 200 bases upstream of UVSX, show homology with previously deduced T4 transcription sites dependent on the T4 transcription factor motA (Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T., and Gold, L. (1988) J. Mol. Biol. 199, 241-258). Analysis of the 3’ end of uvsXRNAs shows that initially most transcripts extend through gene 40 and 41, although Eone-fourth end just past UVSX (within gene 40). Later, more of the uusXmessages are monocistronic, having 5‘ ends close to the gene (200 and 55 bases upstream) and having the 3‘ end within gene 40. Thus, during infection the level of 41 RNA is lowered relative to UUSX message. Mapping of RNA expressed from an UVSX-40-41 plasmid in an unin- fected cell gives 5‘ ends 700, 450, and 55 bases up- stream of UUSX, i.e. positions different from those dur- ing T4 infection. This indicates that infection signifi- cantly changes the 5‘ ends for UUSX RNA, either by altering transcription initiation or RNA processing sites. In contrast, the majority of the UVSX RNAs ex- pressed by plasmid in the uninfected cell do end at the stop mapped during infection. Thus, the host alone can produce this 3‘ end.

Throughout infection, bacteriophage T4 uses the host RNA polymerase to transcribe its DNA, but, nevertheless, phage gene expression is developmentally regulated (for reviews, see Rabussay (1983) and Brody et al. (1983)). Three broad classes of phage gene products, immediate early, middle, and late, have been identified (Saker et al., 1970; O’Farrell and Gold, 1973; Young et al., 1980). Immediate early RNA is seen in the absence of phage protein synthesis, suggesting that this tran- scription initiates at sequences similar to Escherichia coli promoters and uses an unmodified RNA polymerase (Brody et al., 1983). However, at least for some T4 genes, good E. coli promoters do not result in significant early transcription

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(Belin et al., 1987; this paper). Within 2 min of infection, middle phage products appear. A model for middle expression (Schmidt et al., 1970) was originally formulated upon the analysis of T4 RNA from the rIIA/rIIB and gene e regions and the finding that middle expression can be inhibited by the addition of chloramphenicol prior to infection (to inhibit phage protein synthesis). In this model middle RNA arises from the action of T4-encoded factors which cause 1) the firing of new promoters and 2) the elongation of upstream transcripts. The need for T4 proteins is supported by the fact that middle expression is perturbed in infections using the T4 mutants motA, mot& and motC (Mattson et al., 1978; Pulitzer et al., 1979; Linder and Skold, 1980; Pulitzer et al., 1985). Furthermore, the RNA polymerase is rapidly modified after infection both by ADP-ribosylation (Rabussay, 1983) and by the binding of a 10-kDa protein (Stevens, 1972; Stevens and Rhoton, 1975; Orsini and Brody, 1988) and a 15-kDa protein (the product of the T4 rpbA gene) (Stevens, 1972; Malik and Goldfarb, 1984; Hsu et al., 1987; Williams et al., 1987). The functions of the motB gene, motC gene, the 10-kDa protein, the 15-kDa protein, and the effect of the chemical modifica- tion are not yet clear. However, transcript analyses of several T4 motA-dependent genes indicate that wild type motA is necessary for middle transcript initiations at promoters hav- ing a -10 Pribnow box sequence and a new (middle) -35 sequence (Gruidl and Mosig, 1986; Guild et al., 1988). This suggests that the motA protein is involved in changing the specificity of RNA polymerase for the new promoter sequence.

The T4 region encompassing the prereplicative genes UUSX and 41 provides a model for examining the expression of T4 early/middle genes. UUSX encodes a 44-kDa protein, the T4 analog of the E. coli recA protein (Yonesaki et al., 1985; Formosa and Alberts, 1986) which is needed for normal re- combination, replication, and repair (for reviews, see Wakem and Ebisuzaki (1981), Bernstein and Wallace (1983), and Mosig (1983)). Gene 41 protein, along with the product of the T4 gene 61, makes up the primase-helicase which both un- winds DNA at the replication fork and synthesizes the pen- taribonucleotides that initiate lagging strand DNA synthesis (Liu and Alberts, 1980, 1981; Nossal, 1980; Venkatesan et al., 1982; Hinton and Nossal, 1987). Gene 40, which lies between UUSX and 41, encodes a product that stimulates head forma- tion (Hsaio and Black, 197813) and may also provide an early immunity function for the phage (Emrich, 1968; Vallei! and de Lapeyrikre, 1975; Obringer et al., 1988). The entire UUSX- 40-41 region must be expressed as early and/or middle genes since uvsX and 41 proteins are needed for normal DNA replication and 40 has been previously characterized as a typical middle product (Hsaio and Black, 1978a). Differential expression of this region is suggested by the higher level of uvsX protein relative to 41 protein during infection (Burke et al., 1983).

14432

Expression of T4 Genes UVSX, 40, and 41 14433

The RNA mapping analyses reported here assign the expression of the UUSX-40-41 region to middle rather than immediate early transcripts. Initially the middle RNAs are mostly polycistronic species which extend from upstream of UUSX into gene 41. As infection proceeds, more of the RNAs end at a specific site just downstream of the UUSX gene. The accompanying paper (Hinton, 1989) demonstrates that the proportion of UUSX transcripts that stop at this site is different upon infection of the E. coli rho mutant, rho026, suggesting that transcription termination factor rho is involved in the regulation of this 3' end.

MATERIALS AND METHODS'

RESULTS AND DISCUSSION

The uusX-40-41 Region Is Expressed as Middle RNA-The correct expression of the bacteriophage T4 genes UUSX and 41 is critical to the phage's development since the products of these genes are directly involved in DNA replication and recombination. Because of the functions of the encoded pro- teins, uusX and 41 RNA must be expressed prereplicatively, i.e. as either immediate early and/or middle RNA. However, the exact timing of the expression of these genes has not previously been established. 40 protein, whose gene lies be- tween UUSX and 41, has been classified as a typical middle product (Hsaio and Black, 1978a).

To determine the temporal expression of the UUSX-40-41 region, Northern analysis was performed using s R N A probe No. 1 (Fig. 1) complementary to the uvsX gene. A burst of UUSX RNA is first seen during middle expression before the start of DNA replication (6 min post-infection at 30 "C, 4 min at 37 "C.) These initial transcripts are heterogeneous species, ranging from ~ 1 0 0 0 to %OOO bases in size (Fig. 2A, lane 4; B, lane 1; C, lanes 2 and 4). Later in infection, toward the end of middle synthesis (10 min at 37 "C, 15 min at 30 T), two discrete UUSX RNAs of ~ 1 6 0 0 and E1400 bases are apparent (Fig. 2B, lane 2; C, lane 3 ) . Thus, as infection proceeds, more of the UUSX RNAs either remaining or being generated consist of discrete transcripts whose sizes are similar to the uvsX gene itself.

The prereplicative UUSX RNAs arise from middle rather than immediate early transcription. Infection of cells pre- treated with chloramphenicol (Fig. 2C, lane 6 ) , a procedure previously used to define immediate early RNAs, results in no detectable UUSX transcripts. The first detection of UUSX RNA occurs by 2 min post-infection a t 30 "C. While this transcription is not seen by Northern analysis (Fig. 2A, lane 5; C, lane 5 ) , the more sensitive nuclease S1 protection exper- iment (see Fig. 4, Miniprint), indicates that a small amount of RNA, having the same 5' ends as that seen at 6 min post- infection), is observable at this time. The 2-min and 6-min RNAs are resistant to the addition of chloramphenicol after infection (Fig. 2 - 4 , lane 3; see Fig. 4, Miniprint). This result is characteristic of T4 middle RNA which is sensitive to the addition of chloramphenicol before infection but becomes resistant 1 to 2 min after infection (for a review, see Brody et al., 1983).

Since there is no detectable immediate early component to UUSX transcription, one might expect the UUSX gene to be expressed poorly when present on a plasmid in an uninfected cell. However, cells containing pDH428, which has the UUSX-

Portions of this paper (including "Materials and Methods," part of "Results," Table 11, and Figs. 3-8) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

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FIG. 1. The T4 uusX-40-41 region and summary of tran- script analyses. The 4430-bp T4 DNA cloned into pDH428 starts at an EcoRI site (T4 map unit 24.3 (Kutter and Ruger, 1983) desig- nated position 1) and includes the genes uusX (1295-2467 (Fujisawa et al., 1985)), 40 (2460-2804 (Hinton, 1989)), 41 (2814-4241 (Nakin- ishi and Alberts, personal communication)), and the COOH terminus of pgt (1-456 (Tomaschewski et al., 1985)). The positions of restric- tion sites referred to in the text are shown. pDH428A3 is deleted between positions 1720 and 2495. For DNA sequence from the end of UUSX to the start of 41, see accompanying paper (Hinton, 1989). Upper arrows show deduced transcripts from the region when present on the plasmid pDH428 or after T4 infection. Circles denote major 5' ends at positions 400 and 1100 for early/middle T4 RNA, 1100 and 1240 for middle/late T4 RNA, and 600, 850, and 1240 for plasmid RNA. Multiple minor 5' ends seen after T4 infection are denoted by the rectangles (0). DNA and RNA probes used for nuclease S1 mapping analyses are shown below the plasmids. Arrows indicate 5' to 3' direction. DNA probes were end labeled; RNA probes were labeled throughout.

40-41 region of T4 (Fig. 1), express a high level of uvsX protein (Hinton and Nossal, 1986). Likewise, Northern analy- sis indicates that a high level of uusX RNA is expressed by pDH428 in an uninfected cell. This RNA, though, differs from the prereplicative species detected during infection. The UUSX RNA from pDH428 consists of three discrete transcripts of ~ 2 1 0 0 , ~ 1 8 0 0 , a n d ~ 1 4 0 0 nucleotides as well as a background of RNAs ranging from ~ 1 0 0 0 to E4000 nucleotides (Fig. 2 A , lane 2; €3, lane 3; C , lane 1 ). The assignment of the discrete transcripts to UUSX is confirmed by an analysis of the in vivo RNA expressed by pDH428A3 (Fig. 2 A , lane 1 ). pDH428A3 was derived from pDH428 by deleting 780 bp2 of DNA at the COOH terminus of the UUSX gene (Fig. 1) (Hinton and Nossal, 1986). Again, three transcripts are seen, but their positions correspond to sizes of ~ 1 3 0 0 , ~ 1 0 0 0 , a n d ~ 7 0 0 nucleotides, i.e. 700-800 bases less than the transcripts observed with pDH428. (The background bands of 16 S and 23 S RNAs correspond to nonspecific hybridization to these abundant ribosomal RNAs and are seen in the control of cells which contain no plasmid (Fig. 2 A , lane 6) . )

5' Ends of Prereplicatiue 2'4 uusX RNA Differ from Those of UUSX RNA from the Plasmid pDH428-Sl nuclease protec- tion experiments using both end-labeled DNA probes or

The abbreviations used are: bp, base pairs; DTT, dithiothreitol; ssDNA, single-stranded DNA.

14434

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FIG. 2. Northern analyses of T4 uusX transcripts. RNAs, separated in denaturing formaldehyde gels, were hybridized to the 32P-labeled UVSX antisense RNA probe No. 1 (represents positions 1255-2480; see Fig. 1). The positions of molecular weight markers (DNA or the ribosomal 16 S and 23 S RNAs) are denoted. In some cases,

A, RNA (2.5 pgllane) isolated by Method I from: pDH428A3/DH1 (lane 1) ; pDH428/DHl (lane 2); DH1 infected cells were treated for the indicated times with chloramphenicol (CAM), 100 pg/ml, immediately before harvesting.

with T4D' a t 30 "C for 6 min plus 5 min chloramphenicol (lane 3) , 6 min (lane 4 ) , 2 min (lane 5); and DH1, no infection (lane 6). B, RNA (3 pgllane) isolated by Method I1 from: DH1 infected with T4D' a t 37 "C for 4 min plus 1 min chloramphenicol (lane 1 ) or 10 min plus 1 min chloramphenicol (lane 2); and pDH428/DHl with 1 min chloramphenicol (lane 3 ) . C, RNA isolated by Method I from: pDH428/DHl with 1 min chloramphenicol (2.6 pg) (lane 1); DH1 infected with T4D' a t 30 "C for 6 min plus 1 min chloramphenicol (2.7 pg) (lane 2); E. coli B infected with T4D' at 30 "C for 15 min plus 1 min chloramphenicol (2.1 pg) (lane 3 ) , 6 min plus 1 min chloramphenicol (2.6 pg) (lane 4 ) , or 2 min plus 1 min chloramphenicol (2.2 pg) (lane 5); E. coli B pretreated with chloramphenicol for 1 min prior to T4D' infection of 5 min (2.1 pg) (lane 6); and E. coli B, no infection with 1 min chloramphenicol (1.9 pg) (lane 7).

TABLE I The 5' ends of UVSX RNA at 400 and 1100 coincide with potential T4 motA-dependent promoter sequences

bP Consensus sequence for motA-dependent promotef (a / t ) (a / t )TGCTT(t /c )A 11-13 TAn n n T Potential promoter starting transcription at 400' G A TGCTT T C 12 T A T ' " Potential promoter starting transcription at 1100' T T TGCTT A A 13 TATAAT

a The consensus sequence was deduced from the initiation sites of 14 motA-dependent transcripts by Guild et al. (1988).

T4 sequence from Tomaschewski et al. (1985). T4 sequence from Fujisawa et al. (1985).

ssRNA probes labeled throughout (Fig. 1) were performed to determine the 5' ends of the UUSX RNA (Figs. 3-8, see Miniprint). These analyses indicate that prereplicative T4 uusX transcripts are heterogeneous, starting at several posi- tions (Fig. 3, lanes 2 and 3; Fig. 4, lanes 2 and 6-10; Fig. 5, lane 3; Fig. 6, lane 3) . Major 5' ends, mapped at positions 2400 and 21100, are consistent with initiations at T4 motA- dependent (middle) promoter sequences (Table I). Position 1100 shows a nearly perfect match to the consensus sequence recently deduced from an analysis of 14 motA-dependent transcription initiation sites (Guild et al., 1988). Besides these major ends, several other minor ends, whose intensities varied from experiment to experiment, are apparent. The origins of the minor bands are not clear but may reflect minor pro- moters, processing, or degradation of the RNA.

The RNA isolated later in infection, toward the end of middle synthesis, also has heterogeneous 5' ends similar to those seen prereplicatively (Fig. 3, lane 4; Fig. 5, lane 4; Fig. 6, lane 4 ) . Again, the major end a t ~ 1 1 0 0 is observed. In addition a cluster of ends at 21240 is enhanced at 10 min

post-infection over that seen at 4 min. Of the major ends seen during T4 infection, only some ends

in the cluster a t ~ 1 2 4 0 correlate with major 5' ends of steady- state RNA from pDH428 in vivo (Fig. 3, lanes 1 and 4; Fig. 6, lanes 2 and 4 ) . Instead, the other major ends of plasmid RNA are mapped to ~ 6 0 0 and ~ 8 5 0 (Fig. 3, lane 1; Fig. 4, lanes 1 and 13). The end at 600 is assigned to initiation by unmodified RNA polymerase at an E. coli promoter since it is observed after in uitro transcription (Hinton and Nossal, 1986; Fig. 4, lanes 3 and 4, this paper). (A minor 5' end corresponding to position 485 (seen after long exposures of the gel in Fig. 4) corresponds with the other in uitro initiation site; the reason for the diminished level of RNA from this start in uiuo is not clear.) Analysis of the DNA sequence upstream of UUSX (positions 1-510 (Tomaschewski et al., 1985) and positions 510 to the start of the gene3 reveals potential E. coli promoters for initiating transcription a t 480 and 580, consistent with the 5' ends mapped to ~ 4 8 5 and 2600. (See Mulligan et al.

R. L. Ellis and D. M. Hinton, unpublished experiments.

Expression of T4 Genes UVSX, 40, and 41 14435

(1984) for description of the computer analysis used.) How- ever, many other possible promoters upstream of UUSX are also found (presumably because T4 is very AT-rich (70%)), and in particular starts at 870 and 1250 (correlating with the in uiuo ends at ~ 8 5 0 a n d ~ 1 2 4 0 ) are predicted. These ends then may reflect initiation sites that are not recognized in the simple i n uitro system. Alternatively, they could correspond to RNA processing sites whose correlation with promoter sequences is coincidental.

This analysis of UUSX RNA from pDH428 in uitro and in uiuo indicates that unmodified RNA polymerase initiates a high level of UUSX transcription. During phage infection, however, no immediate early expression from the promoter(s) recognized by unmodified polymerase is detected. The pres- ence of hydroxymethylated, glucosylated cytosines in phage T4 DNA or the fact that ADP-ribosylation of the RNA polymerase occurs immediately after infection (Rabussay, 1983) may account for this discrepancy. I t is also possible that the strong promoter at position 600 is active but is very sensitive to phage mechanisms that shut down immediate early transcription. Interestingly, the region upstream of UUSX has been recently assigned to a new T4 gene starting at position 621 (Ellis and Hinton, 1989). Transient immediate early expression from position 600, which might be important for the expression of this gene, would not have been detected by this analysis.

A Major 3' End of UusX RNA Maps Just Downstream of the UUSX Gene-Nuclease S1 analyses (Figs. 7 and 8; Mini- print) map a single, major 3' end to position 2525 in both RNA from infected cells and cells containing pDH428. This position is 60 bases downstream of the uusX gene and im- mediately after a GC-rich hairpin (see also Hinton, 1989). Quantitation of the amount of RNA stopped a t position 2525 relative to that extending into gene 41 indicates that in the strain DH1, one-fourth of the RNA is stopped 4 min after infection, but by 10 min, three-fourths are stopped. Steady- state RNA from cells containing pDH428 is similar to the later time of infection (approximately two-thirds stopped). Thus, as infection proceeds, the level of uusX RNA increases relative to that of gene 41. Similarly, the level of uvsX protein is greater than that of 41 protein during middle synthesis (Burke et al., 1983), and pDH428/DH1 cells express a low level of 41 protein relative to uvsX (Hinton and Nossal, 1986). However, other factors may also down-regulate the level of 41 protein, since the amount of 41 protein expressed by pDH428 is severalfold lower than that of uvsX protein.

These studies indicate that the major 3' end at position 2525 can be generated by the host alone. The end at 2525 is not detected after in vitro transcription using purified RNA polymerase (Fig. 7, lane 10; Fig. 8, lane 9 ) , suggesting that factor-dependent transcription termination or processing is responsible for generating this stop. Evidence for the involve- ment of one host protein, transcription termination factor rho, in the generation of this 3' end is presented in the accompanying paper (Hinton, 1989).

Acknowledgments-I am grateful to Ed Morgan and Marlene Bel- fort for advice on RNA isolations, to M. Nakanishi and B. Alberts for communication of unpublished results, to Nancy Nossal, Sue Garges, and Reed Wickner for helpful discussions, and to Helen Jenerick for typing this manuscript.

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TRANSCRIPT ANALYSES OF M E e - 4 0 - 4 1 REGION OF BACTERIOPHAGE T4: CHANCES 1N THE RNA AS INFECTION PROCEEDS

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