Rimas Nivinskas

T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis.

The consensus sequence of T4 early promoters differs in length, sequence and degree of conservation from that of Escherichia coli sigma(70) promoters. The enzyme interacting with these promoters, and transcribing the T4 genome, is native host RNA polymerase, which is increasingly modified by the phage-encoded ADP-ribosyltransferase, Alt. T4 early transcription is a very active process, possibly out-competing host transcription. The much stronger T4 promoters enhance viral transcription by a factor of at least two and the Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two. To address the question of which promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E. coli promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensus. Second, mutations were introduced into the highly conserved regions of the T4 early promoter, P8.1. The co-occurrence of the promoter-encoding plasmid pKWIII and vector pTKRI, which expresses Alt in E. coli, constitutes a test system that allows comparison of the transcriptional activities of phage and bacterial promoters, in the presence of native, or alternatively ADP-ribosylated RNA polymerase. Results reveal that T4 early promoters exhibit a bipartite structure, capable of strong interaction with both types of RNA polymerase. The -10, -16, -42 and -52 regions are important for transcript initiation with the native polymerase. To facilitate acceleration of transcription, the ADP-ribosylated enzyme requires not only the integrity of the -10, -16 and -35 regions, but also that of position -33, and even more importantly, maintenance of the upstream promoter element at position -42. The latter positions introduced into the E. coli Ptac promoter render this mutant promoter responsive to Alt-ADP-ribosylated RNA polymerase, like T4 early promoters.

ModA and ModB, two ADP-ribosyltransferases encoded by bacteriophage T4: catalytic properties and mutation analysis.

Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Alt- and ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.

Twelve new MotA-dependent middle promoters of bacteriophage T4: Consensus sequence revised

Bacteriophage T4 middle-mode transcription requires Escherichia coli RNA polymerase, phage-encoded transcriptional activator MotA and co-activator AsiA that form a complex at a middle promoter DNA. T4 middle promoters have been defined by a consensus sequence deduced from the list of 14 middle promoters identified in earlier studies. To date, 33 middle promoters have been mapped on the T4 genome. Of these, 12 contain differences even at the highly conserved positions of the consensus sequence. In the T4 prereplicative gene cluster between genes e and rpbA, we have identified 12 new middle promoters, most of which contain differences from the consensus sequence deduced previously. Analysis of base conservation in the different sequence positions of new middle promoters, as well as those identified previously, revealed some new features of middle T4 promoters. We propose to define these promoters by a MotA box (a/t)(a/t)(a/t)TGCTTtA centred at the position -30, the sequence TAtaAT centred at -10 relative to the transcriptional start site, and the spacer region of 12(+/-1) base-pairs between them.

An internal AUU codon initiates a smaller peptide encoded by bacteriophage T4 baseplate gene 26

SummaryBacteriophage T4 baseplate gene 26 codes for two in-frame overlapping peptides with identical C-terminal regions. By site-directed mutagenesis we have now determined that an internal AUU, codon 114 of gene 26, is used as the initiation codon for the synthesis of a smaller peptide (gp26*). Thus gene 26* gives rise to a peptide of 95 amino acid residues with an Mr of 10873, while the complete gene 26 encodes a peptide of 208 amino acid residues of Mr 23 880. Expression of gene 26* is shown to depend on the RNA secondary structure in the translational initiation region of this gene.

Gene rIII is the nearest downstream neighbour of bacteriophage T4 gene 31.

The nucleotide sequence of the 2218-bp T4 DNA fragment encompassing gene 31 and five complete open reading frames (ORFs) is presented. We show here that one of these ORFs, ORF31.-1, located downstream from gene 31, is the rIII gene. The position of the gene was established by comparison with the sequences of the rIII gene mutants, r67, rES40 and rBB9. The ORF corresponding to the rIII gene encodes a basic protein of 82 amino acids with an M(r) of 9323 and a pI of 9.28. According to the Chou and Fasman [Adv. Enzymol. 47 (1978) 45-148] secondary structure prediction, the rIII protein has a relatively high helical content. In addition, discrepancies with the overlapping sequences determined by other authors in this region are indicated.

The sequences of gene rIII of bacteriophage T4 and its mutants

The nucleotide sequences of bacteriophage T4 gene rIII, from six different rIII mutants, have been determined. We show that the mutations r67, rES35 and rES40 cause basic amino acid changes in the rIII protein, while the mutations rBB9, rCR28 and rCP24 cause chain termination.

Middle promoters constitute the most abundant and diverse class of promoters in bacteriophage T4.

The temporally regulated transcription program of bacteriophage T4 relies upon the sequential utilization of three classes of promoters: early, middle and late. Here we show that middle promoters constitute perhaps the largest and the most diverse class of T4 promoters. In addition to 45 T4 middle promoters known to date, we mapped 13 new promoters, 10 of which deviate from the consensus MotA box, with some of them having no obvious match to it. So, 30 promoters of 58 identified now deviate from the consensus sequence deduced previously. In spite of the differences in their sequences, the in vivo activities of these T4 middle promoters were demonstrated to be dependent on both activators, MotA and AsiA. Traditionally, the MotA box was restricted to a 9 bp sequence with the highly conserved motif TGCTT. New logo based on the sequences of currently known middle promoters supports the conclusion that the consensus MotA box is comprised of 10 bp with the highly conserved central motif GCT. However, some apparently good matches to the consensus of middle promoters do not produce transcripts either in vivo or in vitro, indicating that the consensus sequence alone does not fully define a middle promoter.

The nucleotide sequence between genes 31 and 30 of bacteriophage T4

The nucleotide sequence of the 2994 bp T4 phage DNA fragment between genes 31 and 30 is presented. The fragment contains 7 complete open reading frames in the direction of early transcription and two early promoters, PE128.6 and PE128.2, which we show to cause difficulties in cloning DNA from this genomic region. Our data complete the nucleotide sequence and the organization of genes in the genomic region between T4 genes 31 and 30.

Non‐canonical RNA arrangement in T4‐even phages: accommodated ribosome binding site at the gene 26‐25 intercistronic junction

Translational initiation region of bacteriophage T4 gene 25 contains three potential Shine and Dalgarno sequences: SD1, SD2 and SD3. Mutational analysis has predicted that an mRNA stem‐loop structure may include SD1 and SD2, bringing the most typical sequence SD3, GAGG, to the initiation codon. Here, we report physical evidence demonstrating that previously predicted mRNA stem‐loop structure indeed exists in vivo during gene 25 expression in T4‐infected Escherichia coli cells. The second mRNA stem‐loop structure is identified 14 nucleotides upstream of the stem‐loop I, while the SD3 sequence, as well as the start codon of the gene, are proved to be within an unfolded stretch of mRNA. Phylogenetic comparison of 38 T4‐like phages reveals that the T‐even and some pseudoT‐even phages evolve a similar structural strategy for the translation initiation of 25, while pseudoT‐even, schizoT‐even and exoT‐even phages use an alternative mRNA arrangement. Taken together, the results indicate that a specific mRNA fold forms the split ribosome binding site at the gene 26‐25 intercistronic junction, which is highly competent in the translational initiation. We conclude that this ribosome binding site has evolved after T‐even diverged from other T4‐like phages. Additionally, we determine that the SD sequence GAGG is most widespread in T4.

Cloning, sequence, and overexpression of bacteriophage T4 gene 51

The nucleotide sequence of the 907-bp XbaI-EcoRV T4 DNA fragment containing gene 51 is presented. The sequence of gene 51 predicts a 249 amino acid peptide with an M(r) of 29387 and a pl of 6.34. We have cloned and overexpressed this gene in the T7 RNA polymerase system. The observed molecular mass of gp51 is in agreement with the sequence data. We also show that the low level of gene 51 expression usually seen is caused by an RNA stem-loop structure in the region between genes 26 and 51. In addition, discrepancies with the sequence published by other authors are indicated.