Transcription of Bacteriophage T4 DNA in vitro: Selective Initiation with Dinucleotides

Abstract

The transcription products of phage T4 DNA in vitro are separated on polyacrylamide gels. The influence of salt, polymerase, triphosphate concentration and glucosylation on the RNA synthesis are shown. Individual transcripts are initiated selectively with dinucleotides and a single triphosphate. This technique allows the prediction of the initiation sequences of several T4 transcripts.The linear and double-stranded DNA of bacteriophage T4 has a molecular weight of 1.19 x lo8 [l], corresponding to approximately 160 000 base pairs. About 100 genes of T4 are identified and localized on a circular map [2,3]. The sequence of the genes is circularly permuted [4,5] and the genome shows 2-3 % redundancy [6]. In place of cytosine T4 DNA contains 5-hydroxymethylcytosine [7] to which glucose is attached in c1 or fi linkage [8,9], rendering the DNA resistant against host restriction enzymes [lo]. In addition 0.35 % of the adenine residues are modified toThroughout phage development T4-specific RNA synthesis remains sensitive to rifampicin [13,14]. Therefore it is assumed that the host enzyme participates in transcription of all T4 mRNA. In vitro, however, the only promoters recognized efficiently by the Escherichiu coli RNA polymerase are those of the immediate early functions [15,16] the RNA of which is found in vivo at about 1.5 min after infection. A modification of the host RNA polymerase and the binding of phage-coded proteins to it then seem to be required in vivo for the transcription of the other classes of T4 mRNA. Among the T4-coded proteins involved in the transcription of the later This paper is dedicated to R. W. Kaplan on the occasion of his 65th birthday.Abbreviations. Abbreviations for nucleotides follows CBN recommendations, Eur. J. Biochem. 15,203-208 (1970); C-T4 DNA, cytosine-containing T4 DNA.Enzymes. RNA polymerase or DNA-dependent RNA polymerase (EC 2.7.7.6); RNase 111 or double-stranded-ribonucleate 3'-oligonucleotidohydrolase (EC 3.1.4.24); EcoRI or endodeoxyribonuclease EcoRI (EC 3.1.4.32).classes of mRNA are the products of the T4 genes mot, 45, 55, 33 and alc [20-251. To study T4 transcription in vitro it is desirable to separate clearly single transcription products. It will be shown in this paper that electrophoresis of T4 transcripts on a polyacrylamide gel results in a pattern of defined RNA bands. The chain lengths of the transcripts range between about 10000 and 450 nucleotides. The influence of template DNA, salt, enzyme and ribonucleoside triphosphate concentration will be investigated. Initiation of T4 transcription with dinucleotides allows the synthesis of single transcripts to be selected for. The sequences of some initiation sites will be predicted by alignment of overlapping trinucleotides stimulating the synthesis of unique transcripts. MATERIALS AND METHODS MaterialsUnlabeled nucleoside triphosphate and polyinosinic acid, (rI)" were purchased from Boehringer

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex

“…The crystal structure of one such bacteriophage T4 β-glucosyltransferase has been determined (Vrielink et al, 1994). Glucosylation of phage DNA was also implicated as having a control function in phage-specific gene expression (Cox and Conway, 1973;Rüger, 1978).…”

Biochemical Characterization of Recombinant β-Glucosyltransferase and Analysis of Global 5-Hydroxymethylcytosine in Unique Genomes

“…1,2 Furthermore, glucosylation is also implicated in phage specific gene expression by influencing transcription. 3−5 …”

Changes in the promoter range of RNA polymerase resulting from bacteriophage T4-induced modification of core enzyme.

“…3. primers (32,34). Of the two promoters of the tRNA region, P1 responds to initiation with UpA and P2 responds to initiation with CpA (34).…”

“…A recently developed technique ofelectrophoretic separation of primary in vitro T4 transcripts (31,32) allows direct analysis of promoter site selection by purified RNA polymerases. Two ofthe in vitro T4 transcripts made by hostpolymerase have been identified as products of the T4 tRNA gene cluster (see Fig.…”