Transcription of the T4 late genes

Abstract

This article reviews the current state of understanding of the regulated transcription of the bacteriophage T4 late genes, with a focus on the underlying biochemical mechanisms, which turn out to be unique to the T4-related family of phages or significantly different from other bacterial systems. The activator of T4 late transcription is the gene 45 protein (gp45), the sliding clamp of the T4 replisome. Gp45 becomes topologically linked to DNA through the action of its clamp-loader, but it is not site-specifically DNA-bound, as other transcriptional activators are. Gp45 facilitates RNA polymerase recruitment to late promoters by interacting with two phage-encoded polymerase subunits: gp33, the co-activator of T4 late transcription; and gp55, the T4 late promoter recognition protein. The emphasis of this account is on the sites and mechanisms of actions of these three proteins, and on their roles in the formation of transcription-ready open T4 late promoter complexes.

Figure 1

The T4 late promoter sequence logo.

Figure 2

Amino acid sequence conservation of gp55. All T4-related phage genomes sequenced to date (see [59], which is a review by Petrov, et al., in this series) contain readily identifiable gp55 homologues [81]. Four segments of sequence conservation can be noted. The central and largest segment 2 allows the distant relationship to domain 2 of σ⁷⁰ to be discerned, primarily through correspondence with σ⁷⁰ conserved segments 2.1 and 2.2 and secondary structure. The presumption that segment 2.4 harbors the late promoter recognition element of gp55 is speculative. Conserved segment 4 is the sliding clamp-binding epitope. Conserved segments 1 and 3 share no recognizable sequence similarity with σ⁷⁰. Whether they correspond functionally with σ segment 1.1/1.2 and 3.1, respectively, is not known. The numbering of residues is continuous for the T4 protein. Amino acid sequences of the T4, RB14 and RB32 proteins are identical; only T4 is listed. RB49 and phi-1 gp55 are also identical except for Q30 (RB49)→E30 (phi-1); only RB49 is listed. A secondary structure prediction from HHpred, with α-helices as cylinders, is shown below the alignment. Vertical lines at the side cluster phages infecting (top to bottom): E. coli (133 was isolated as an Acinetobacter phage); Aeromonas species; and Vibrio species. The more divergent S-PM2 protein is the only representative of the completely sequenced cyanobacterial phages that has been included for this presentation. (The cyanobacterial RNAPs constitute a separate clade in the phylogeny of the multisubunit enzymes, as do the archaeal RNAPs and the individual eukaryotic nuclear RNAPs I-V.)

Figure 3

Bacterial RNAP holoenzyme. A. The Thermus aquaticus RNAP holoenzyme. The β (pink), β" (pale green), α₂ (yellow, orange; without their C-terminal domains) and ω (cyan) subunits are identified, and the β subunit flap (red), which is the attachment site of σ domain 4 and gp33, as well as the β" coiled-coil (green), which is the docking site of σ domain 2 and gp55, are emphasized. σ domains 1.2, 2, 3 and 4 (dark blue) are identified. B. The same, with σ removed (i.e., RNAP core, but with the coordinates of the holoenzyme) (Adapted from [26]).

Figure 4

The limited sequence conservation of gp33. The presentation of the sequence alignment follows Figure 2. Amino acid sequences of the T4 and RB14 proteins are identical; RB32 gp33 differs only by E50→K; only the T4 protein is listed. RB43 and RB16 gp33 are identical and only RB43 is listed. A secondary structure prediction from HHpred is shown below the alignment.

Figure 5

A composite partial molecular model of the sliding clamp docking on an RNAP:promoter complex. The structure of the RB69 sliding clamp [47] has been docked against a Taq RNAP holoenzyme fork junction promoter DNA complex [25]. Evidence from site-specific DNA-protein photochemical cross-linking and DNA footprinting [34] specifies that the sliding clamp abuts RNAP. Gp33 is placed in the model in accordance with the recent determination of its structure in complex with the E. coli β flap and DRII (amino acids 831-1057) by K-A.F. Twist and S.A. Darst [43][K-A.F. Twist, P. Deighan, S. Nechaev, A. Hochschild, E.P. Geiduschek & S.A. Darst, in preparation] and a complete structural model of E. coli RNAP based on a combination of approaches [82]. Placement of the C-end of gp33 in proximity to DNA is consistent with evidence from site-specific DNA-protein cross-linking [34]. The rotational orientation of gp45 is arbitrary, but is likely to be constrained by the interacting RNAP surface and also by the short tether to gp33. The location of the C-end of gp33 on the sliding clamp in the T4 late promoter complex is not known; a C-terminal 11-mer of phage RB69 DNA polymerase from the structure in [47] has not been removed and is barely visible, but its relevance to the late promoter complex is unclear, as discussed in the text. Residues 44-123 of gp55, comprising its RNAP core- and DNA-biding sites, have been modeled based on homology with σ⁷⁰ domain 2 [26] and docked onto the β" subunit coiled-coil. Colors of components are indicated in the Figure. (Images provided by K.-A. Twist and S.A. Darst and reproduced with their permission.)

Figure 6

A simplified 2-step model for kinetic analysis of the formation of initiation-ready open promoter complexes.