A combination of structural approaches yields a complete atomic model of the highly biochemically characterized Escherichia coli RNA polymerase, enabling fuller exploitation of E. coli as a model for understanding transcription.

The biochemical characterization of the bacterial transcription cycle has been greatly facilitated by the production and characterization of targeted RNA polymerase (RNAP) mutants. Traditionally, RNAP preparations containing mutant subunits have been produced by reconstitution of denatured RNAP subunits, a process that is undesirable for biophysical and structural studies. Although schemes that afford the production of in vivo-assembled, recombinant RNAP containing amino acid substitutions, insertions, or deletions in either the monomeric b or b 0 subunits have been developed, there is no such system for the production of in vivo-assembled, recombinant RNAP with mutations in the homodimeric a-subunits. Here, we demonstrate a strategy to generate in vivo-assembled, recombinant RNAP preparations free of the a C-terminal domain. Furthermore, we describe a modification of this approach that would permit the purification of in vivoassembled, recombinant RNAP containing any a-subunit variant, including those variants that are lethal. Finally, we propose that these related approaches can be extended to generate in vivoassembled, recombinant variants of other protein complexes containing homomultimers for biochemical, biophysical, and structural analyses.

A reverse-genetics approach has been used to probe the mechanism underlying immune escape for influenza A virus-specific CD8 + T cells responding to the immunodominant D b NP 366 epitope. Engineered viruses with a substitution at a critical residue (position 6, P6M) all evaded recognition by WT D b NP 366 -specific CD8 + T cells, but only the NPM6I and NPM6T mutants altered the topography of a key residue (His155) in the MHC class I binding site. Following infection with the engineered NPM6I and NPM6T influenza viruses, both mutations were associated with a substantial "hole" in the naïve T-cell receptor repertoire, characterized by very limited T-cell receptor diversity and minimal primary responses to the NPM6I and NPM6T epitopes. Surprisingly, following respiratory challenge with a serologically distinct influenza virus carrying the same mutation, preemptive immunization against these escape variants led to the generation of secondary CD8 + T-cell responses that were comparable in magnitude to those found for the WT NP epitope. Consequently, it might be possible to generate broadly protective T-cell immunity against commonly occurring virus escape mutants. If this is generally true for RNA viruses (like HIV, hepatitis C virus, and influenza) that show high mutation rates, priming against predicted mutants before an initial encounter could function to prevent the emergence of escape variants in infected hosts. That process could be a step toward preserving immune control of particularly persistent RNA viruses and may be worth considering for future vaccine strategies.

Activated transcription of the bacteriophage T4 late genes, which is coupled to concurrent DNA replication, is accomplished by an initiation complex containing the host RNA polymerase associated with two phage-encoded proteins, gp55 (the basal promoter specificity factor) and gp33 (the coactivator), as well as the DNAmounted sliding-clamp processivity factor of the phage T4 replisome (gp45, the activator). We have determined the 3.0 Å-resolution X-ray crystal structure of gp33 complexed with its RNA polymerase binding determinant, the β-flap domain. Like domain 4 of the promoter specificity σ factor (σ 4 ), gp33 interacts with RNA polymerase primarily by clamping onto the helix at the tip of the β-flap domain. Nevertheless, gp33 and σ 4 are not structurally related. The gp33/β-flap structure, combined with biochemical, biophysical, and structural information, allows us to generate a structural model of the T4 late promoter initiation complex. The model predicts protein/protein interactions within the complex that explain the presence of conserved patches of surface-exposed residues on gp33, and provides a structural framework for interpreting and designing future experiments to functionally characterize the complex.| Gp33 | replication-coupled gene expression | X-ray crystallography T ranscription initiation in bacteria depends on the RNA polymerase (RNAP) catalytic core (subunit composition α 2 ββ 0 ω) and the promoter specificity subunit σ, which combine to create the RNAP holoenzyme. The σ subunit recruits RNAP to promoters through sequence-specific interactions with two conserved hexameric DNA sequence motifs, the −10 and −35 promoter elements. The −10 element is recognized by structural domain 2 of σ (σ 2 ), positioned on the clamp helices of the RNAP β′-subunit, while the −35 element is recognized by σ 4 positioned on the flap-tip-helix (FTH) of the RNAP β-subunit flap domain (1, 2). Transcription from weak promoters may be modulated by activators that typically bind to specific DNA sequences, or operators, located upstream of the −10∕ − 35 promoter elements, and stabilize the initiation complex through protein/protein interactions directly with the RNAP holoenzyme, often with the α-subunit

Exposure to naturally occurring variants of herpesviruses in clinical settings can have a dramatic impact on anti-viral immunity. Here we have evaluated the molecular imprint of variant peptide-MHC complexes on the T-cell repertoire during human cytomegalovirus (CMV) infection and demonstrate that primary co-infection with genetic variants of CMV was coincident with development of strain-specific T-cell immunity followed by emergence of cross-reactive virus-specific T-cells. Cross-reactive CMV-specific T cells exhibited a highly conserved public T cell repertoire, while T cells directed towards specific genetic variants displayed oligoclonal repertoires, unique to each individual. T cell recognition foot–print and pMHC-I structural analyses revealed that the cross-reactive T cells accommodate alterations in the pMHC complex with a broader foot-print focussing on the core of the peptide epitope. These findings provide novel molecular insight into how infection with naturally occurring genetic variants of persistent human herpesviruses imprints on the evolution of the anti-viral T-cell repertoire.