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Membrane protein insertion and topogenesis generally occur at the Sec61 translocon in the endoplasmic reticulum membrane. During this process, membrane spanning segments may adopt two distinct orientations with either their N- or C-terminus translocated into the ER lumen. While different topogenic determinants in membrane proteins, such as flanking charges, polypeptide folding, and hydrophobicity, have been identified, it is not well understood how the translocon and/or associated components decode them. Here we present evidence that the translocon-associated protein (TRAP) complex is involved in membrane protein topogenesis in vivo. Small interfering RNA (siRNA)-mediated silencing of the TRAP complex in HeLa cells enhanced the topology effect of mutating the flanking charges of a signal-anchor, but not of increasing signal hydrophobicity. The results suggest a role of the TRAP complex in moderating the ‘positive-inside’ rule.

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.

The Escherichia coli T4 bacteriophage uses two glycosyltransferases to glucosylate and thus protect its DNA: the retaining alpha-glucosyltransferase (AGT) and the inverting beta-glucosyltransferase (BGT). They glucosylate 5-hydroxymethyl cytosine (5-HMC) bases of duplex DNA using UDP-glucose as the sugar donor to form an alpha-glucosidic linkage and a beta-glucosidic linkage, respectively. Five structures of AGT have been determined: a binary complex with the UDP product and four ternary complexes with UDP or UDP-glucose and oligonucleotides containing an A:G, HMU:G (hydroxymethyl uracyl) or AP:G (apurinic/apyrimidinic) mismatch at the target base-pair. AGT adopts the GT-B fold, one of the two folds known for GTs. However, while the sugar donor binding mode is classical for a GT-B enzyme, the sugar acceptor binding mode is unexpected and breaks the established consensus: AGT is the first GT-B enzyme that predominantly binds both the sugar donor and acceptor to the C-terminal domain. Its active site pocket is highly similar to four retaining GT-B glycosyltransferases (trehalose-6-phosphate synthase, glycogen synthase, glycogen and maltodextrin phosphorylases) strongly suggesting a common evolutionary origin and catalytic mechanism for these enzymes. Structure-guided mutagenesis and kinetic analysis do not permit identification of a nucleophile residue responsible for a glycosyl-enzyme intermediate for the classical double displacement mechanism. Interestingly, the DNA structures reveal partially flipped-out bases. They provide evidence for a passive role of AGT in the base-flipping mechanism and for its specific recognition of the acceptor base.