Isolation and characterization of conditional lethal mutations in the mot gene of bacteriophage T4

Mattson, T.; Houwe, Griet Van; Epstein, Richard H.

doi:10.1016/0022-2836(78)90058-x

Cited by 65 publications

(37 citation statements)

References 39 publications

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“…In T2 the gene 59-gene 32 intergenic sequence contains an intercalated middle and late promoter that both transcribe monocistronic gene 32 mRNAs (47). The same classes of promoters transcribe gene 32 in T4, but in this case these promoters are embedded within the inserted gene 32.1 sequence (5,47,49,74). Since the same classes of promoters transcribe gene 32 in both of the T-even versions of this locus, we had assumed that some strong regulatory constraint imposed this conservation.…”

Section: Discussionmentioning

confidence: 99%

Snapshot of the Genome of the Pseudo-T-Even Bacteriophage RB49

et al. 2002

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RB49 is a virulent bacteriophage that infects Escherichia coli. Its virion morphology is indistinguishable from the well-known T-even phage T4, but DNA hybridization indicated that it was phylogenetically distant from T4 and thus it was classified as a pseudo-T-even phage. To further characterize RB49, we randomly sequenced small fragments corresponding to about 20% of the Ϸ170-kb genome. Most of these nucleotide sequences lacked sufficient homology to T4 to be detected in an NCBI BlastN analysis. However, when translated, about 70% of them encoded proteins with homology to T4 proteins. Among these sequences were the numerous components of the virion and the phage DNA replication apparatus. Mapping the RB49 genes revealed that many of them had the same relative order found in the T4 genome. The complete nucleotide sequence was determined for the two regions of RB49 genome that contain most of the genes involved in DNA replication. This sequencing revealed that RB49 has homologues of all the essential T4 replication genes, but, as expected, their sequences diverged considerably from their T4 homologues. Many of the nonessential T4 genes are absent from RB49 and have been replaced by unknown sequences. The intergenic sequences of RB49 are less conserved than the coding sequences, and in at least some cases, RB49 has evolved alternative regulatory strategies. For example, an analysis of transcription in RB49 revealed a simpler pattern of regulation than in T4, with only two, rather than three, classes of temporally controlled promoters. These results indicate that RB49 and T4 have diverged substantially from their last common ancestor. The different T4-type phages appear to contain a set of common genes that can be exploited differently, by means of plasticity in the regulatory sequences and the precise choice of a large group of facultative genes.Approximately 170 bacteriophages with morphologies similar to T4 have been identified (1). These T4-like phages have been isolated on a wide range of bacterial hosts that grow in diverse environments (1, 2, 72). T4, the type phage of this family, is probably the best-understood virulent phage. Its genome has been entirely sequenced, and its life cycle is extremely well understood; however, until recently little was known about all the other T4-type phages.Genomic hybridization and PCR analysis revealed that the T4-type phages vary considerably in their distance from T4 (54,62,72). Based on such data and limited sequence analysis, we can distinguish subgroups of the T4 type (54,62,72). The T-even subgroup shares considerable nucleic acid sequence homology with T4; for example, quantitative hybridization between the genomes of T2, T4, and T6 indicates that more than 90% of their sequences are nearly identical (18). As a result of their close relationship to T4, the T-even genomes can usually be analyzed and sequenced using PCR primers based on the T4 sequence (62, 71, 72). Although such comparisons confirmed that most of the T4-type phages were very close to T4, a few of th...

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Section: Discussionmentioning

confidence: 99%

Snapshot of the Genome of the Pseudo-T-Even Bacteriophage RB49

et al. 2002

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“…Wild-type T4D ϩ , T4 amG1 (motA) (25), or T4 amS22 (asiA) (33) were used for infections. The E. coli B strains TabG (39), which is restrictive for T4 motA(Am) and motA(Ts) growth, and NapIV (30) have been described previously.…”

Section: Methodsmentioning

confidence: 99%

“…Amber (Am) and temperature sensitive (Ts) mutants exhibit a delay in DNA synthesis and form small plaques (25,33) while motA and asiA deletions are lethal (5,37). Analyses of RNA isolated from motA(Am) mutant infections reveal that these mutants are leaky; although transcription from many middle promoters is nearly eliminated, for particular promoters, a low level of RNA can still be detected (11,24,49,51).…”

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A Mutation within the β Subunit ofEscherichia coliRNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters

2010

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During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. Middle genes, whose expression begins about 1 min postinfection, are transcribed both from the extension of early RNA into middle genes and by the activation of T4 middle promoters. Middle-promoter activation requires the T4 transcriptional activator MotA and coactivator AsiA, which are known to interact with 70 , the specificity subunit of RNA polymerase. T4 motA amber [motA(Am)] or asiA(Am) phage grows poorly in wild-type E. coli. However, previous work has found that T4 motA(Am)does not grow in the E. coli mutant strain TabG. We show here that the RNA polymerase in TabG contains two mutations within its ␤-subunit gene: rpoB(E835K) and rpoB(G1249D). We find that the G1249D mutation is responsible for restricting the growth of either T4 motA(Am)or asiA(Am) and for impairing transcription from MotA/AsiA-activated middle promoters in vivo. With one exception, transcription from tested T4 early promoters is either unaffected or, in some cases, even increases, and there is no significant growth phenotype for the rpoB(E835K G1249D) strain in the absence of T4 infection. In reported structures of thermophilic RNA polymerase, the G1249 residue is located immediately adjacent to a hydrophobic pocket, called the switch 3 loop. This loop is thought to aid in the separation of the RNA from the DNA-RNA hybrid as RNA enters the RNA exit channel. Our results suggest that the presence of MotA and AsiA may impair the function of this loop or that this portion of the ␤ subunit may influence interactions among MotA, AsiA, and RNA polymerase.Bacterial RNA polymerase (RNAP) is a highly conserved enzyme that shares sequence and structural homology with multisubunit polymerases from single-celled archaea to multicellular eukaryotes (19,55). In bacteria, an RNAP core consisting of five subunits (␣ 1 , ␣ 2 , ␤, ␤Ј, and ) combines with a specificity factor, , to form holoenzyme (13). A primary factor, such as 70 of Escherichia coli, is used for promoter recognition during exponential growth; alternate factors are needed under specific growth conditions or times of stress (10, 34). In addition, proteins that bind to RNAP and/or the DNA can influence the activity of polymerase (2,4,16,18,42).During infection of E. coli, bacteriophage T4 relies on the host transcriptional machinery for transcription from phage early, middle, and late promoters (reviewed in references 16, 26, and 54). Immediately after infection, synthesis of early RNA is driven by T4 early promoters, which contain strong matches to Ϫ10 and Ϫ35 DNA elements recognized by 70 . Expression of T4 middle genes is delayed, commencing after 1 to 2 min. Middle RNA is generated both from early promoters, whose transcripts extend into middle genes, and from specific middle promoters. While middle promoters contain the 70 -dependent Ϫ10 element, they have a Ϫ30 element (MotA box) rather than the Ϫ35 element recogniz...

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“…Wild-type T4D ϩ phage was from B. Stitt, Temple University, Philadelphia, Pa. The temperature-sensitive T4 motA mutant tsG1 and the motA amber mutant amG1 (25,26) were from K. Kreuzer. Complementation tests with these mutants were performed as described previously (10).…”

Section: Methodsmentioning

confidence: 99%

“…Although several T4 motA mutants have been isolated and characterized (7,17,18,20,25,26,39,47), analyses of the sequence changes present in these mutations reveal that all retain at least the first 59 amino acids of the wild type-MotA sequence (33,45,47). We describe the isolation and characterization of an N-terminal mutation of motA, mot21, in which the first 8 codons of the wild-type motA gene are replaced with 11 different codons.…”

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An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription

Gerber

Hinton

1996

J Bacteriol

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The bacteriophage T4 MotA protein is a transcriptional activator of T4-modified host RNA polymerase and is required for activation of the middle class of T4 promoters. MotA alone binds to the ؊30 region of T4 middle promoters, a region that contains the MotA box consensus sequence [(t/a)(t/a)TGCTT(t/c)A]. We report the isolation and characterization of a protein designated Mot21, in which the first 8 codons of the wild-type motA sequence have been replaced with 11 different codons. In gel retardation assays, Mot21 and MotA bind DNA containing the T4 middle promoter P uvsX similarly, and the proteins yield similar footprints on P uvsX . However, Mot21 is severely defective in the activation of transcription. On native protein gels, a new protein species is seen after incubation of the 70 subunit of RNA polymerase and wild-type MotA protein, suggesting a direct protein-protein contact between MotA and 70 . Mot21 fails to form this complex, suggesting that this interaction is necessary for transcriptional activation and that the Mot21 defect arises because Mot21 cannot form this contact like the wild-type activator.Transcription of the middle class of bacteriophage T4 genes requires T4-modified host RNA polymerase and a transcriptional activator, the product of the T4 motA gene (reviewed in reference 43). The modification to the polymerase required for MotA-dependent transcription has been identified as the T4 AsiA protein (13, 32), a factor that binds tightly to 70 (41, 42). A mutation in the motA gene was discovered because the mutant phage fails to give normal patterns of T4 prereplicative protein synthesis (25). Subsequent work demonstrated that this abnormal protein pattern observed during a T4 motA mutant infection occurs because the middle (MotA-dependent) class of promoters does not become active (6). Middle promoters are characterized by an excellent match to the Ϫ10 consensus sequence for 70 , the specificity subunit of RNA polymerase, but they lack a recognizable 70 Ϫ35 sequence. Instead they share a 9-bp sequence, a MotA box, centered 30 bp upstream of the start of transcription (2, 6). The MotA protein binds to this sequence and is required for transcription initiating at T4 middle promoters by T4-modified polymerase in vitro (10,24,37). Recent results indicate that while T4-modified polymerase can bind a middle promoter in the absence of the activator, MotA protein is needed to form the open complex (12).The motA gene encodes a protein of 211 amino acids (45). Although several T4 motA mutants have been isolated and characterized (7,17,18,20,25,26,39,47), analyses of the sequence changes present in these mutations reveal that all retain at least the first 59 amino acids of the wild type-MotA sequence (33,45,47). We describe the isolation and characterization of an N-terminal mutation of motA, mot21, in which the first 8 codons of the wild-type motA gene are replaced with 11 different codons. The wild-type MotA and Mot21 proteins bind similarly to the T4 middle promoter P uvsX , but Mot21 fails to activ...

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Isolation and characterization of conditional lethal mutations in the mot gene of bacteriophage T4

Cited by 65 publications

References 39 publications

Snapshot of the Genome of the Pseudo-T-Even Bacteriophage RB49

Snapshot of the Genome of the Pseudo-T-Even Bacteriophage RB49

A Mutation within the β Subunit ofEscherichia coliRNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters

An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription

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