The number of CTD heptad repeats increased substantially in the ancestor of all Metazoa (31 to 44 repeats).
This was accompanied by the appearance of repeats with lysine at position seven (K CTD.) Thus, we conclude that CTD repeat length markedly increased with the origin of animals, and the distal repeats gained lysine residues and expanded further in different animal lineages.
Disruption of this mechanism interfered with the expression of two growth-factor-induced genes regulated by polymerase pausing, but did not influence expression or polymerase occupancy at two non-paused genes [ residues, and thus the potential for RPB1 CTD acetylation, arose with animal multicellularity during an expansion in the overall number of CTD repeats in Metazoa.
Our phylogenetic analysis further showed that p300/KAT3B, the acetyltransferase that modifies the RPB1 CTD, was present at the appearance K-containing repeats.
Given the association between polymerase pausing and ac RPB1, the potential to acetylate these residues may have enabled tighter control of gene expression as animals grew in complexity and diversified.
Phylogenetic tree of eukaryotic species considered in our analysis organized by approximate divergence estimates.The largest subunit, called RPB1, is unique to RNA polymerase II and is involved in its catalytic activity.The C-terminal domain (CTD) of RPB1 is essential for the proper regulation of RNA polymerase II  and contains a protein-protein interaction surface for cofactors involved in the regulation of transcription initiation, elongation and RNA processing—highly specialized functions that determine the speed and reliability of the polymerase enzyme traversing a gene during transcription .However, there is considerable diversity in the number and sequence of the repeats across eukaryotic clades, and the complexity of the CTD is roughly correlated with developmental complexity in animal, plant, and fungal multicellular lineages [), where the repeats lack lysines.This modification, performed by the acetyltransferase (KAT) enzyme p300/KAT3B, which is absent in yeast, distinctly marks promoter-proximally paused polymerases.In this schematic of the RPB1 CTD for two species of yeast and human, consensus heptad repeats (YSPTSPS) are colored dark gray; repeats with a lysine at position 7 are colored red; and all other non-consensus repeats are in white. Human, mouse, and zebrafish were selected as representative vertebrate species, based on their phylogenetic placement and sequence data quality.