Marc Uzan

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

Bacteriophage T4 early promoters are utilized immediately after infection and are abruptly turned off 2–3 min later (at 30°C) when the middle promoters are activated. The viral early protein AsiA has been suspected to bring about this transcriptional switch: not only does it activate transcription at middle promoters in vivo and in vitro but it also shows potent anti‐σ70 activity in vitro, suggesting that it is responsible for the shut‐off of early transcription. We show here that after infection with a phage deleted for the asiA gene the inhibition of early transcription occurs to the same extent and with the same kinetics as in a wild‐type infection. Thus, another AsiA‐independent circuit efficiently turns off early transcription. The association of a mutation in asiA with a mutation in mod, rpbA, motA or motB has no effect on the inhibition of early promoters, showing that none of these phage‐encoded transcriptional regulators is necessary for AsiA‐independent shut‐off. It is not known whether AsiA is able to inhibit early promoters in vivo, but host transcription is strongly inhibited in vivo upon induction of AsiA from a multicopy plasmid.

Nucleotide sequence and control of transcription of the bacteriophage T4 motA regulatory gene

A 2116bp segment of the bacteriophage T4 genome encompassing the motA regulatory gene has been sequenced. In addition to motA, five open reading frames were identified in the direction of early transcription. The motA gene encodes a basic protein of 211 amino acids with a predicted molecular weight of 23559. Measurements of the rate of transcription of motA showed that the promoter of this gene is turned off after only 2 min of T4 development. This early promoter presents a structure which is richer in information than that of a classical constitutive Escherichia coli promoter. In addition to containing conserved sequences centred at ‐10 and ‐35, this promoter shares extensive homologies with other subgroups of early promoters in regions centred at +3 and at ‐55. We discuss the possible role of these different sequence determinants.

Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation

Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10 - 15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC) and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit); and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes) of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context.

Chapter 2 RNA Processing and Decay in Bacteriophage T4

Bacteriophage T4 is the archetype of virulent phage. It has evolved very efficient strategies to subvert host functions to its benefit and to impose the expression of its genome. T4 utilizes a combination of host and phage-encoded RNases and factors to degrade its mRNAs in a stage-dependent manner. The host endonuclease RNase E is used throughout the phage development. The sequence-specific, T4-encoded RegB endoribonuclease functions in association with the ribosomal protein S1 to functionally inactivate early transcripts and expedite their degradation. T4 polynucleotide kinase plays a role in this process. Later, the viral factor Dmd protects middle and late mRNAs from degradation by the host RNase LS. T4 codes for a set of eight tRNAs and two small, stable RNA of unknown function that may contribute to phage virulence. Their maturation is assured by host enzymes, but one phage factor, Cef, is required for the biogenesis of some of them. The tRNA gene cluster also codes for a homing DNA endonuclease, SegB, responsible for spreading the tRNA genes to other T4-related phage.

Phage T4 early promoters are resistant to inhibition by the anti-sigma factor AsiA

Phage T4 early promoters are transcribed in vivo and in vitro by the Escherichia coli RNA polymerase holoenzyme Eσ70. We studied in vitro the effects of the T4 anti‐σ70 factor AsiA on the activity of several T4 early promoters. In single‐round transcription, promoters motB, denV, mrh.2, motA wild type and UP element‐deleted motA are strongly resistant to inhibition by AsiA. The α‐C‐terminal domain of Eσ70 is crucial to this resistance. DNase I footprinting of Eσ70 and Eσ70AsiA on motA and mrh.2 shows extended contacts between the holoenzyme with or without AsiA and upstream regions of these promoters. A TG → TC mutation of the extended −10 motif in the motA UP element‐deleted promoter strongly increases susceptibility to inhibition by AsiA, but has no effect on the motA wild‐type promoter: either the UP element or the extended −10 site confers resistance to AsiA. Potassium permanganate reactivity shows that the two structure elements are not equivalent: with AsiA, the motA UP element‐deleted promoter opens more slowly whereas the motA TC promoter opens like the wild type. Changes in UV laser photoreactivity at position +4 on variants of motA reveal an analogous distinction in the roles of the extended −10 and UP promoter elements.

New System for Positive Selection of Recombinant Plasmids and Dual Expression in Yeast and Bacteria Based on the Restriction Ribonuclease RegB

By coupling the toxic restriction endoribonuclease RegB, from the bacteriophage T4, to the prokaryotic T7 and the eukaryotic GAL1 promoters, we constructed a two‐function plasmid called pTOXR‐1. This plasmid is a zero‐background cloning vector. It allows an efficient positive selection of recombinant plasmids without the need to completely digest, dephosphorylate, or purify the vector prior to the ligation step. The pTOXR‐1 positive selection system requires no special Escherichia coli strains, no special culture media, and no addition of inducer to the selective plates. In addition, since this vector carries all signals required for both prokaryotic and eukaryotic expression, it allows the overproduction of heterologous proteins, fused to a polyhistidine tag, in the bacterium E. coli and in the yeast Saccharomyces cerevisiae from a single plasmid. Hence, this vector may be a useful time‐saving tool for one‐step cloning and versatile protein expression in bacteria and yeast.