Proteins are constructed from chains of amino acids and often have modular regions known as domains. One end of every protein chain is the carboxy-terminal, or C-terminal, end. A domain found at this location in some proteins is the Carboxy-Terminal Domain (CTD), a specialized region that acts as a hub for cellular activities. The CTD is a feature of proteins central to gene expression, serving as a platform for complex molecular interactions and coordination.
The Unique Structure of the CTD
The most studied example of a Carboxy-Terminal Domain belongs to the largest subunit of an enzyme called RNA polymerase II, which is responsible for transcribing genes. This CTD has a defining feature: it is made up of a long series of repeating amino acid sequences. In humans and mice, this sequence is repeated 52 times, creating a long, tail-like extension from the main body of the enzyme. The repeating sequence is a heptapeptide, consisting of seven amino acids with the consensus sequence Tyrosine-Serine-Proline-Threonine-Serine-Proline-Serine (Y-S-P-T-S-P-S).
This repetitive architecture gives the CTD a unique physical property. It is considered an intrinsically disordered region, which means it does not have a stable, defined three-dimensional structure like most other protein domains. Instead, it is flexible and mobile, capable of changing its shape and interacting with a wide variety of other molecules. This structural fluidity is directly related to its ability to function as a dynamic regulatory platform.
Regulation Through Modification
The CTD’s function is controlled by post-translational modification, where chemical groups are added to the protein after its creation. For the CTD, the primary modification is phosphorylation, the addition of phosphate groups. The repeating heptapeptide sequence is rich in amino acids that can be phosphorylated, specifically the two serines, the threonine, and the tyrosine. This allows for an immense number of possible modification patterns along the length of the CTD.
This system of modifications is often referred to as the “CTD code.” Different enzymes, known as kinases, add phosphate groups at specific positions, while other enzymes, called phosphatases, remove them. The specific pattern of phosphorylation at any given moment determines the CTD’s shape and which other proteins it can bind to. For example, phosphorylation at the fifth amino acid in the repeat (Serine-5) is a signal during the early phases of gene transcription, while phosphorylation at the second position (Serine-2) is associated with later stages.
Coordinating Gene Expression
The CTD of RNA polymerase II acts as a coordinator for converting a gene’s DNA into messenger RNA (mRNA). It functions as a mobile scaffold that recruits the machinery for each stage of transcription. This process ensures the steps of gene expression happen in the correct order, physically linking transcription with mRNA processing.
During initiation, the first stage of transcription, the CTD is largely unphosphorylated. This state helps RNA polymerase II and other factors assemble at the start of a gene. Once the polymerase is in position, progression into the elongation phase requires a change in the CTD’s modification state. Kinase enzymes add phosphate groups, particularly at the Serine-5 position, which helps the polymerase begin synthesizing the mRNA strand.
As RNA polymerase II moves along the DNA during elongation, the CTD’s phosphorylation pattern continues to evolve, recruiting a new set of factors. The phosphorylated tail becomes a binding platform for enzymes that process the new mRNA molecule as it is made. This includes the machinery for 5′ capping, the components of the spliceosome, and factors for polyadenylation, which adds a tail of adenine bases to signal termination.
Significance in Health and Disease
Errors in the CTD’s structure or regulation are linked to a variety of human diseases due to its central role in controlling gene expression. These issues can arise from mutations in the gene that codes for the CTD or from malfunctions in the kinase and phosphatase enzymes that manage the CTD code.
Dysregulation of CTD function is a factor in several types of cancer. In some malignancies, the enzymes that phosphorylate the CTD are overactive, leading to uncontrolled transcription of genes that promote cell growth. For instance, the kinase CDK7, which phosphorylates Serine-5, is a target of interest in cancer therapy because inhibiting it can disrupt the transcription of cancer-promoting genes. Mutations affecting CTD phosphatases have also been observed in various cancers.
Beyond cancer, defects in CTD-mediated transcription are implicated in other conditions. Some neurological and developmental disorders are associated with mutations in transcription factors that interact with the CTD, disrupting the proper expression of genes necessary for normal development. The link between the CTD and mRNA processing means subtle changes can affect which versions of proteins are made, a process important in the nervous system.