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We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1-PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N-hydroxysuccinimidobiotin (NHS-biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described. Five regions in the PB1 monomer showed altered reactivity to NHS-biotin when compared with the [PB1-PA] heterodimer. Mutational analysis of residues in two such regions-at K265 and K481 of PB1, which were about three- and twofold, respectively, less accessible to biotinylation in the PB1-PA heterodimer compared with the PB1 monomer, demonstrated that both K265 and K481 were crucial for polymerase function. This novel assay of quantitative profiling of biotinylation patterns (Q-POP assay) highlights likely conformational changes at important functional sites, as observed here for PB1, and may provide information on protein-protein interaction interfaces. The Q-POP assay should be a generally applicable approach and may detect novel functional sites suitable for targeting by drugs.

The genome of influenza A virus consists of eight negative-stranded RNA segments which have partially complementary non-coding terminal sequences. Previous transcription studies of the virion RNA promoter in vitro have shown that the 5' terminus forms an integral part of the promoter and an 'RNA-fork' model has been proposed for the initiation of transcription. According to this model part of the promoter is formed by an RNA-duplex which involves complementary residues 10 to 1 2 of the 3' end and residues 11' to 13' of the 5' end. With a reverse genetics system, based on the chloramphenicol acetyltransferase (CAT) gene, we have now tested this part of the promoter in vivo. Single mutations of the conserved residues at positions 11 and 12 of the 3' terminus and at positions 12' and 13' of the 5' terminus abolished promoter activity. The introduction of complementary mutations into both termini partially restored activity. On the other hand, mutations at positions 10 of the 3' terminus and 11' of the 5' terminus inhibited activity independently of whether a base-pair was formed or not. Thus, at these positions, the nature of the residues is apparently more important than their ability to form base-pairs. These results extend our previous virion 'RNA-fork' model and are consistent with in vitro findings that the 5' terminus is involved in the initiation of transcription.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.