Transcription is an essential step in the expression of genetic information. This process depends on protein complex of multisubunit RNA polymerases that are exceptionally conserved among all cellular organisms. These enzymes together with eukaryotic RNA-dependent RNA polymerases involved in gene silencing form a monophyletic protein family whose members contain two double-ψ β-barrel structural motifs in their active center. This family also includes a group of mainly in silico predicted non-canonical DNA-dependent RNA polymerases which differ from multisubunit RNA polymerases in reduced composition. Putative non-canonical RNA polymerase consisting of two subunits is also encoded by cytoplasmic linear plasmids of the yeast Kluyveromyces lactis and highly likely transcribes genes of these plasmids. Characterization of a unique transcription machinery of Kluyveromyces lactis plasmids with major emphasis on non-canonical RNA polymerase has become the aim of this work. Bioinformatic analysis in silico was used to examine the evidence leading to an assumption of existence of specific RNA polymerase. Subsequent genetic and biochemical methods were used for: 1) production of putative RNA polymerase subunits in several expression systems; 2) testing interaction between several components of transcription...

Bacterial type I restriction-modification systems are composed of three different subunits: one HsdS subunit is required for identification of target sequence and anchoring the enzyme complex on DNA; two HsdM subunits in the methyl-transferase complex serve for host genome modification accomplishing a protective function against self-degradation; two HsdR (or motor) subunits house ATP-dependent translocation and consequent cleavage of double stranded DNA activities. The crystal structure of the 120 kDa HsdR subunit of the Type I restriction-modification system EcoR124I in complex with ATP was recently reported. HsdR is organized into four approximately globular structural domains in a nearly square-planar arrangement: the N-terminal endonuclease domain, the RecA-like helicase domains 1 and 2 and the C-terminal helical domain. The near-planar arrangement of globular domains creates prominent grooves between each domain pair. The two helicase-like domains form a canonical helicase cleft in which double-stranded B-form DNA can be accommodated without steric clash. The helical domain, probably involved in complex assembly, exhibits only a few specific interactions with helicase 2 domain. Molecular mechanism of dsDNA translocation, cleavage and ATP hydrolysis has not been yet structurally investigated. Here we propose a translocation cycle of the restriction-modification system EcoR124I based on analysis of available crystal structures of superfamily 2 helicases, strutural modeling and complementary biochemical characterization of mutations introduced in sites potentially inportant for translocation in the HsdR motor subunit. Also a role of the extended region of the helicase motif III in ATPase activity of EcoR124I was probed.