The rZZA Gene of Bacteriophage T 4 . 11 . Regulation of Its Messenger RNA Synthesis

When the rZZ genes are first introduced into cells which had been previously infected by T4 phage deleted for these genes, the kinetics of synthesis of rZZA and rZZB RNA are rapid and identical. We show that this rapid synthesis depends on a functional motA gene for rZZB, but not for rZZA, RNA synthesis. By primer-extension mapping of T4 messenger RNA, we find three promoters close to the rZZA gene. One of them is an early promoter just before the rZZA. 1 gene; it is used under all conditions tested. Another is in the coding portion of the rZZA.1 gene; it is weak, primarily because of a 19-bp spacing between the -10 and -35 elements, and its use is stimulated by T 4 functions. The third is a motA-dependent (middle) promoter which has an unusual CCCGCTT box at -33. We present results which suggest that none of these promoters is likely to be the site at which the motB and motC gene products exercise their major influence on rZZA RNA synthesis. A LTHOUGH the rZZA gene of bacteriophage T 4 has played an important role in the history of genetics, its role in the T 4 life cycle is still obscure [see the companion study (DAEGELEN and BRODY 1990); and SINGER, SHINEDLING and GOLD 1983)l. The control of biosynthesis of the rZZA gene product is also not fully understood. The rZZA gene is part of an early transcription unit (a transcription unit for which initiation of RNA synthesis requires no T4 proteins). Moreover, there is a p-sensitive termination site between this early promoter and the rZZA gene (SEDEROFF, BOLLE and EPSTEIN 1971; SEDEROFF et al. 1971; DAEGELEN and BRODY 1976; CARUSO et al. 1979; PULITZER, COPPO and CARUSO 1979; YOUNG AND CRONE MENARD 1980; DAEGELEN, D’AUBENTONCARAFA and BRODY 1982; THERMES and BRODY 1984). This picture of rZZA as a “classic” delayed-early gene of T 4 (BRODY, RABUSSAY and HALL 1983) is clearly insufficient to explain a number of experimental results. When T 4 development is allowed to take place for several minutes before the rZZ genes are introduced into infected cells by superinfection, the kinetics of rZZA RNA synthesis are not of the delayed-early type; rather, rZZA and rZZB RNA appear with the same rapid kinetics (DAEGELEN and BRODY 1976). This suggests that a promoter exists close to the rZZA gene, as close, in fact, as is the middle rZZZ3 promoter to the rZZB gene. This latter promoter determines an RNA start only 122 nucleotides upstream of the ATG of rZZB Biotechnologie, INRA Domaine de Vilvert, 78350 Joiny en Josas, France. Genetics 1 2 5 249-260 (June, 1990) I Present address: Group SystPmes ParallPles et Biologie, Institut de (SCHMIDT et al. 1970; PRIBNOW et al. 1981; GUILD et al. 1988). Such an rZZA promoter was found by SELZER et al. 1978; 198 1). They showed that a DNA fragment containing the beginning of the rZZA gene and extending partially into the upstream gene 60 contains a promoter which drives rZZA RNA synthesis when cloned into plasmid pBR313. The question remains why this promoter does not produce a detectable immediate-early transcript when it is part of an injected T4 DNA molecule. The expression of rZZA RNA in the middle mode is also unclear. Elimination of the motA gene product, a positive activator of middle-mode transcription (BRODY, RABUSSAY and HALL 1983; GUILD et al. 1988), does not eliminate rZZA RNA synthesis (DAEGELEN, D’AUBENTON-CARAFA and BRODY 1982; PuLITZER, COLOMBO and CIARAMELLA 1985). In fact, it leads to overproduction of the rIIA protein (MATTSON, RICHARDSON and GOODIN 1974; MATTSON, VAN HOUWE and EPSTEIN 1978). However, simultaneously eliminating motA function and deleting two other loci of T 4 (motB and motC) does lead to a reduction of rZZA RNA synthesis (PULITZER, COLOMBO and CIARAMELLA 1985). The roles of motB and motC are not clear, although the motB protein has been identified and has been shown to be localized in the same prereplicative DNA-protein complex as the motA protein (UZAN et al. 1985) . Finally, rZZA RNA is known to be translationally repressed by the T 4 regA protein (WIBERG and KARAM 1983; MILLER et al. 1987; WINTER et al. 1987; also, see DAEGELEN and BRODY 1990). Armed with the DNA sequence of the rZZA gene, we have reinvestigated rZZA expression. We have iden250 P. Daegelen and E. Brody