Sequence and characterization of the bacteriophage T4 comC alpha gene product, a possible transcription antitermination factor

Abstract

We have sequenced a 1,340-bp region of the bacteriophage T4 DNA spanning the comCa gene, a gene which has been implicated in transcription antitermination. We show that comCav, identified unambiguously by sequencing several missense and nonsense mutations within the gene, codes for an acidic polypeptide of 141 residues, with a predicted molecular weight of 16,680. We have identified its product on one-and two-dimensional gel systems and found that it migrates abnormally as a protein with a molecular weight of 22,000. One of the missense mutations (comCa803) is a glycine-to-arginine change, and the resulting protein exhibits a substantially faster electrophoretic mobility. The ComCa protein appears immediately after infection. Its rate of synthesis is maximum around 2 to 3 min postinfection (at 37°C) and then starts to decrease slowly. Some residual biosynthesis is still detectable during the late period of phage development.The notion that termination and antitermination mechanisms may be operating in bacteriophage T4 development initially came from the observation of a strong polarity on early transcription when infection is carried out in the absence of translation. In vivo experiments that used inhibitors of protein synthesis, as well as in vitro studies, have shown that this polarity is due to the action of the host transcription terminator p (2). The question of whether translation of phage RNA is required just to mask potential rho-dependent termination sites and/or to allow the synthesis of viral antitermination proteins could not be answered on the basis of experiments that used inhibitors of protein synthesis. Furthermore, analysis of this problem is complicated by the fact that many genes, transcribed by elongation of transcripts initiated at distal early promoters, are also transcribed from proximal promoters (the middle promoters) activated soon after infection (2,10,13,24,30,45,46).Another set of experiments showing the involvement of p in T4 development derives from the isolation of rho mutants unable to support growth of T4 wild-type phage. They were called tabC (5, 40, 41) or hdf (38). It has been suggested that the altered p proteins made in these mutants are insensitive to T4-induced antitermination factors (31, 38). Thus, these mutations can be considered as producing a "super-rho" phenotype. Further support for this view is provided by the observation that one of these rho mutations (rhoO26; also called nusDO26) (37,38) was shown to impede the antitermination activity of XN, specifically at rho-dependent terminators (8).Phage mutants able to grow on tabC/hdf rho host mutants were isolated. These compensatory mutations, called comC or goF, were mapped mainly in two places on the chromosome: upstream of gene 39, in a nonessential region of the T4 genome, and between genes 55 and e (5,7,17,36,38,42 involved in replication and in head assembly, respectively. Infection of a super-rho strain (hdf/rhoO26) with wild-type phage leads to an increased proportion of RNA ending at a specific site within g...

Effects on mRNA degradation by Escherichia coli transcription termination factor Rho and pBR322 copy number control protein rop

“…Our results, in agreement with a proposal made by Sanson & Uzan (1992), suggest that the T4 GoF1 mutant protein, which partly restores the ability of T4 to propagate in rho(nusD) cells, may also be an RNA-binding protein. This proposal is consistent with the results presented by Hinton (1989), that the level of T4 gene 41 RNA is higher in rho026 cells infected with T4goF1 compared to T4 .…”

Bacteriophage T4 polynucleotide kinase triggers degradation of mRNAs

“…Thus, until sufficient PNK has accumulated to trigger their degradation by RNases G and E, these relatively stable early transcripts should produce their encoded proteins long after early promoters have shut down (minutes 2-3 postinfection) (10,21). In agreement with this hypothesis, we have previously observed that the biosynthesis of ComCα, one of the proteins encoded by the cef mRNA, persists until the onset of the late period, although with a decreasing rate of synthesis (23). Fig.…”

The bacteriophage T4 anti‐sigma factor AsiA is not necessary for the inhibition of early promoters in vivo

“…Efficient mechanisms thus switch off ongoing transcription each time a new developmental phase starts. In all cases, translational repression mechanisms are superimposed on transcriptional inhibitory mechanisms (Young et al ., 1980; Uzan et al ., 1990; Sanson and Uzan, 1993; Cowan et al ., 1994; Kutter et al ., 1994a; Miller et al ., 1994; Sanson and Uzan, 1995).…”

“…To date, the activity of seven early promoters has been monitored in the course of T4 infection and all seven are turned off 2–3 min after infection at 30°C (Broida and Abelson, 1985; Uzan et al ., 1990; Sanson and Uzan, 1993; B. Sanson and M. Uzan, unpublished), making likely the notion that all T4 early promoters (≈ 40) undergo the same negative regulation very soon after infection. One of the most plausible candidates for early promoter inhibition is the AsiA protein.…”