The middle and late genes are transcribed by the host RNAP modified by the products of two early phage genes 7, 19, 20, 21. Genes of the P23-45 phage were previously divided into three temporal expression classes: early, middle, and late 7.

These findings provide novel insight into the striking versatility and evolvability of the transcription machinery.Ī putative RNA polymerase is a part of the tail tape measure protein

We show that despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. In this work, we characterize unique virion- and non-virion RNAPs of Thermus thermophilus bacteriophage P23-45 and compare them to other phage and cellular two-barrel RNAPs. Comprehensive identification and study of highly diverged viral two-barrel RNAPs can be expected to yield insights into the mechanisms and evolution of transcription. However, the possibility that some single-subunit viral RNAPs predate cellular enzymes, i.e., are relics of ancient RNAPs antedating the LUCA, cannot be discarded. The low sequence similarity among the viral two-barrel RNAPs suggests their origin through independent acquisition from cells, followed by simplification of the domain architecture accompanied by extensive divergence. Typically, viral two-barrel RNAPs are organized more simply than their cellular counterparts, lacking some or even all small subunits 10, 13, 15, 16, 17 or consisting of a single polypeptide with two DPBB domains fused 14, 18. Many bacterial and eukaryotic viruses with double-stranded DNA genomes in the realms Duplodnaviria and Varidnaviria encode homologs of cellular RNAPs (collectively denoted “two-barrel RNAPs”) 7, 8, 9, 10, 11, 12, 13, 14. Although more complex, the archaeal and eukaryotic RNAPs contain homologs of each bacterial RNAP subunit, implying that the Last Universal Common Ancestor of cellular organisms (LUCA) already encoded a multisubunit RNAP from which the extant enzymes in all the domains of life are derived 1. The DPBB domain of the β′ subunit contains a conserved amino acid motif DxDxD, where the three aspartates coordinate Mg 2+ ions required for the catalysis of RNA synthesis. The active site is located at the interface of two double-psi β-barrel (DPBB) domains formed by the large subunits. Bacterial RNAPs are composed of two large catalytic subunits β and β′, a dimer of α subunits that serves as an assembly platform for the large subunits, and the ω subunit 4, 5, 6. Genes of cellular organisms are transcribed by multisubunit DNA-dependent RNA polymerases (RNAPs), which are conserved in all three domains of life 1, 2, 3. Our findings demonstrate striking adaptability of RNAPs that can be attained within a single virus species. Structure and sequence comparisons of the P23-45 RNAPs with other RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. By contrast, the virion RNAP adopts a unique flat structure without a clamp. The non-virion RNAP has a crab-claw-like architecture. We report the crystal structures of both P23-45 RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes.

Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail.

Biorad western blot protein ladder