NMR studies on the structure of an influenza virus RNA promoter, a hemimethylated DNA, and a putative nucleoid-associated protein in bacteria

Abstract

The influenza A virus, a severe pandemic pathogen, has a segmented RNA genome consisting of eight single-stranded RNA molecules. The 5’ and 3’ ends of each RNA segment recognized by the influenza A virus RNA-dependent RNA polymerase direct both transcription and replication of the virus’s RNA genome. Promoter binding by the viral RNA polymerase and formation of an active open complex are prerequisites for viral replication and proliferation. The viral promoter has a significant dynamic nature and an unusual displacement of an adenosine that forms a novel (A-A)ㆍU motif and a C-A mismatch stacked in a helix. The characterized structural features of the promoter imply that the specificity of polymerase binding results from an internal RNA loop. In addition, an unexpected bending (46±10°) near the initiation site suggests the existence of a promoter recognition mechanism similar to that of DNA-dependent RNA polymerase and a possible regulatory function for the terminal structure during open complex formation. DNA methylation occurs in most organisms from bacteria to mammals and provides a mechanism for epigenetic control of a variety of cellular processes. In Escherichia coli, most of the N6 positions in adenines found in the sequence GATC are methylated by DNA adenine methyltransferase. After DNA replication, the GATC sites exist transiently in the hemimethylated state, and the specific recognition of these hemimethylated GATC sites is essential for several processes, including sequestration of the site of replication initiation by the SeqA protein, strand discrimination in DNA mismatch repair by the MutH protein, and transcription of several genes. The N6-methyl of hemimethylated GATC sites undergoes a slow trans-cis interconversion without disruption of the double helix. The release of tightly bound cation from hemimethylated DNA explains the instability of this structure. In addition, quantitative structural analysis revealed that hemimethylated DNA has unusual...